Vitamin D autocrine signaling - illustrated tutorial

Robin Whittle rw@firstpr.com.au  2021-09-25
(First established 2020-11-23.)

../ To the main page of this site.

For an overview of vitamin D and the immune system, please see: https://vitamindstopscovid.info/05-mds/ .

Almost all of the numerous (hundreds) of functions of the vitamin D compounds in the body are through autocrine (within the one cell) signaling, and the simple extension of this which is paracrine signaling (signaling to nearby cells).  All the immune system functions of vitamin D work in these ways.

This is unrelated to the one hormonal function of vitamin D, which is a very low, but tightly regulated, concentration of circulating 1,25OHD to control calcium-bone metabolism.

Yet most people - including many doctors - are not familiar with autocrine or paracrine signaling.  To understand vitamin D in general - and especially to understand why population-wide vitamin D repletion targeting 40 to 60ng/ml 25OHD vitamin D blood levels (100 to 150nmol/L) is the key to improving health - we need a good understanding of vitamin D based autocrine signaling. 

As far as I know, all this is correct, since it is based on some earlier text which met with the approval of a senior vitamin D researcher - but see the terminology section concerning this page's use of autocrine to include what may be more specifically referred to as intracrine signaling.   If you spot any errors or can suggest any improvements, please let me know.


Contents

#00-term
Notes on terminology.  Read this first to reduce later confusion.  Added 2021-09-25.
#01-compounds D3 cholecalciferol, 25OHD calcifediol and 1,25OHD calcitriol.
#02-nothorm Hormonal 1,25OHD for calcium-bone metabolism is at a much lower level than the 1,25OHD generated as an autocrine agent and/or paracrine agent in immune and other cells, so the hormonal 1,25OHD levels, which are quite stable, have no significant effect on the autocrine and paracrine signaling systems of numerous types of cell.
#03-minlev Numerous reasons why we should aim for at least 40ng/ml (100nmol/L) 25OHD blood levels.
#04-quraishi 2014 research which indicates we should aim for at least 55 or 60ng/ml 25OHD blood levels.
#05-auto Description of autocrine signaling with two examples from research articles, the second of which is directly relevant to severe COVID-19.  Paracrine signaling is easily understood as an extension of this to nearby cells.


#00-term

Confused and confusing terminology, including: "Vitamin D" "hormone" and "autocrine" and "intracrine" signaling


Update 2021-09-25:  Here is my best attempt at untangling some contradictory and/or divergent and at least confusing terminological problems.  The following includes my own value judgments on how particular terms should be used, and how they are sometimes misused.

The term Vitamin D is generally and properly used to refer collectively to the three compounds best known in mammalian biology:
Sometimes "vitamin D" is used to refer just to D3, and "vitamin D metabolites" to the other to compounds, as well as to other compounds not mentioned here such as those which result from the breakdown of any of the above three compounds

The vitamin D receptor AKA VDR.  According to the Wikipedia page [WP] an alternative term for this is calcitriol receptor.  I have never seen this term, but it is substantially more correct and would ideally be widely used.  However, I suspect that we are stuck with the current terminology, and "VDR" is short and distinctive.

D3 and 25-hydroxyvitamin D have a very low affinity for the VDR, so it is generally wrong to think of it as a receptor for these compounds.  1,25-dihydroxyvitamin D (calcitriol) has a far greater affinity for the VDR, but the lower affinities should not be forgotten in scenarios where there is little 1,25-dihydroxyvitamin but very high levels of D3 and 25-hydroxyvitamin D. 

There are an alternative set of three compounds to those above, based on Vitamin D2 ergocalciferol which is produced industrially and is not a normal part of mammalian biology.  Here is a rare diagram showing both the molecular differences and the carbon numbers, from this site.



D2 and its 25- and 1,25 hydroxylated forms has no advantages over the D3 compounds, so generally I don't discuss them.  For some obscure historical reasons doctors in the USA were only able to prescribe D2, and often still do - I have been told that this is no longer mandated.

The term vitamin is questionable.  From a book chapter cited here , where the milk may be fortified with D2.

Although it is classified as a vitamin because of the nature of the discovery process, vitamin D in actual fact should not be considered a true vitamin for the following reasons. First and foremost, vitamin D utilized by higher organisms can be formed in the epidermis of skin by photolysis of 7-dehydrocholesterol, an intermediate in cholesterol biosynthesis. Second, vitamin D is nearly absent from the food supply. Vitamin D is found in fish liver oils, some fatty fish, and in egg yolk but is not found in virtually all plant materials, in skeletal meats, seeds, fruits, and vegetables. In fact, very little is found in an expected source, milk. We consider milk an important supply of vitamin D in many countries, primarily because it is fortified by the addition of vitamin D.

It is common to find the phrase "vitamin D is a hormone" (Google Scholar 711 hits) as if this is technically more accurate than the questionable term "vitamin" - or as if this important group of compounds deserves additional gravitas not conferred by "vitamin".  For additional oomph, there is also "Vitamin D is a secosteroid hormone" (328 hits). 

Secosteroid refers to breaking the B ring of a steroid molecule [WP] which is the only way known in nature and industry to make the vitamin D compounds - and this break can only be achieved with a very narrow set of wavelengths in the UV-B spectrum.  It so happens that the Sun produces these, in the top fraction of a percent of its frequency range (the short wavelength end of its spectrum) and that if the Sun is high enough in the sky, appreciable amounts of these wavelengths pass through the atmosphere to reach the Earth's surface.  The exact wavelengths are a matter of research and debate, but are in the 293 to 297 nanometre range.  See Industrial Aspects of Vitamin D Arnold L. Hirsch, 2011: https://sci-hub.se/10.1016/B978-0-12-381978-9.10006-X .

It is common for the term vitamin D to be used both for referring collectively to the compounds listed above, and potentially other related compounds, which I think is a correct use of the term, and to use it when the author is actually referring to one of these compounds, but does not refer to it specifically.  The reader is left to infer exactly which compound is meant, which is a terrible mistake in this already difficult field.

I was happy to find a 2004 article concerning common terminological mistakes:

Why “Vitamin D” is not a hormone, and not a synonym for  1,25-dihydroxy-vitamin D, its analogs or deltanoids
Reinhold Vieth
J. Steroid Biochemistry and Molecular Biology 2004-06-30
https://www.sciencedirect.com/science/article/abs/pii/S0960076004000858 (Paywalled.)
https://sci-hub.se/10.1016/j.jsbmb.2004.03.037

"Deltanoid" seems to refer to analogues of "vitamin D" - non-natural molecules which mimic the behaviour of naturally occurring vitamin D compounds.  A company of this name worked on these, but the term is obscure and probably not used in 2021.

Abstract: Official nutrition committee reports in both North America and Europe now state that Vitamin D is more of a hormone than a nutrient.  These statements are wrong, and do not reflect the definitions of either vitamin or hormone. Researchers often compound the problem by referring to calcitriol or other deltanoids as "Vitamin D". These things have serious consequences:

(1) The literature is burdened by an ongoing confusion that presumes that the reader will somehow “know” what the writer refers to by "Vitamin D". 

(2) Medical practitioners not familiar with the ambiguities administer Vitamin D inappropriately when calcitriol or a deltanoid analog would be correct, or vice versa. 

(3) Attempts to promote Vitamin D nutrition are hindered by alarmist responses justifiably associated with the widespread administration of any hormone

Vitamin D is a vitamin in the truest sense of the word, because "insufficient amounts in the diet may cause deficiency diseases". The term prohormone is not relevant to the Vitamin D system, but 25-hydroxy-Vitamin D (calcidiol) is appropriately described as a prehormone, i.e. a glandular secretory product, having little or no inherent biologic potency, that is converted peripherally to an active hormone.


But even here there are problems, since with sufficient UV-B skin exposure, there is no need for D3 in food.  In 2004 it was reasonable to think of 1,25-dihydroxyvitamin D as a "hormone" (the last word of the abstract) because it was not widely known that it also has numerous non-hormonal roles in autocrine / intracrine / paracrine signaling.

As best I can tell, the term hormone [WP] originally meant signaling molecule with the unstated assumption that the signaling path was from one type of cell (or at least an organ somewhere) to one or more other cell types (such as in one or more other organs).  

The signaling is the conveyance of information by way of the level (concentration) of the compound.  This is detected by the recipient cells. 

In plants (which lack organs) the long distance, within the plant itself, transport of the hormone molecules is by the plant's fluid transport system.  In vertebrates,  the signal is conveyed by the level (concentration) of the substance in the bloodstream and potentially the CSF (Cerebrospinal Fluid [WP]) and interstitial fluid [WP]. 

Unfortunately the term hormone is sometimes also applied to any signaling molecule - including those which operate over short distances such as within cells and to nearby cells. 

Today, the only proper use of the term hormone in vertebrates is as described above, and not to refer to signaling molecules which operate within cells or between nearby cells. 

Endocrine signaling [WP] is hormonal signaling for vertebrates.  The Wikipedia page concentrates on this, but also includes in the field of endocrinology three other signaling modalities, while specifically excluding neurotransmitter signaling: autocrine, paracrine and juxtacrine.  However, I am not at all sure that endocrinologists in general, or those who write endocrinology textbooks, are really interested in these fields, since they have nothing in common with hormonal signaling.

Juxtacrine signaling [WP] involves cells in direct contact, including with junctions.  This is somewhat like synaptic communication with neurotransmitters, but is not related to the nervous system.  The vitamin D compounds are not involved in this.

Here are the number of Google search hits within the English Wikipedia for:

autocrine 532
paracrine 659
intracrine 206

From Hector F DeLuca's 2014 History of the discovery of vitamin D and its metabolites  PMC3899558

What we now know as D3 cholecalciferol was isolated and its structure determined in 1937.  Until the 1960s (I recall) the only way of assaying a substance for its D3 content was to feed varying amounts of it to baby rats, to see which ones developed rickets.  From this, the IU (International Unit) was developed - the amount the baby rat needed per day to avoid rickets.  This was later found to be 1/40,000,000 of a gram.  The impressively named International Unit is a very small quantity indeed, and average daily D3 needs such as 1/8000 gram 5000IU appear to many people to be scarily large.  This article mentions autocrine and paracrine, but not intracrine, signaling.

25-hydroxyvitamin D was discovered in 1968 and 1,25-dihydroxyvitamin D in 1971.

Here is my current (2021-09-25) understanding of these three interrelated terms and concepts.  The Wikipedia references are for general information and an indication of current usage, not to indicate that these definitions are authoritative.  I am not sure that there is a single source of authoritative definitions of scientific terms such as these, since usage and definitions change as research progresses.

Intracrine signaling, in which the compound acts as an "intracrine agent" or "intracine", involves the intracrine agent being synthesised in the cell and being sensed by a receptor inside the same cell.  This term is currently actively used, such as in this 2020 article of which Professor Martin Hewison is one of the co-authors.  He and his colleagues led the research into auto/intra/paracrine signaling in the late 2000s.

The Wikipedia page https://en.wikipedia.org/wiki/Intracrine refers to these compounds as hormones which is incompatible with the long distance signaling definition of "hormone" mentioned above.  A Google search for intracrinology reveals references going back to 1991 Intracrinology by Fernand Labrie pubmed/1838082/ sci-hub , which contains the following diagram. 



The Wikipedia page https://en.wikipedia.org/wiki/Autocrine_signaling refers to the "hormone or chemical messenger" binding to receptors on the cell which produced it.  This implies outward facing receptors on the cell membrane.

Autocrine signaling, by the above WP's and diagram's narrow definition, involves a cell producing a compound which leaves the cell and then activates receptors located in the same cell's membrane, from the outside of the cell.   As far as I know, there is no such pattern with vitamin D compounds, where the signaling agent would be 1,25-dihydroxyvitamin D, since the VDR is an intracellular receptor. 

In this definition, what would stop this process also causing the activation of nearby cells of the same and or different type?  That would be paracrine signaling.

Autocrine signaling, by a broader definition, which ignores the location of the receptor, includes both intracrine signaling (above) and (non-existent for vitamin D compounds) narrowly defined autocrine signaling.   This means, that for vitamin D compounds this use of "autocrine" signaling is a synonym for paracrine signaling.

I guess this has happened for reasons including people not being fussed about exactly where the receptor is located.  The signaling system is intracellular - from one set of events in the cell to cause another set of events in the same cell.

Paracrine signaling [WP] broadly means a paracrine agent being generated in a cell of type X and diffusing to nearby cells where it alters the behavior of other cells, of type Y and/or perhaps of type X.  The above diagram defines this as involving receptors facing outwards from the cell membrane, but I think the term also applies to the paracrine agent diffusing into the recipient cell, where it binds to receptors in the cytosol.  I assume this is the case for 1,25-dihydroxyvitamin D paracrine signaling, since I have never read of VDRs being located on the cell membrane.

This is quite messy.  Arguably the most important article ever written on the etiology of severe COVID-19:

An autocrine Vitamin D-driven Th1 shutdown program can be exploited for COVID-19
Reuben McGregor, (22 others), Majid Kazemian and Behdad Afzali. 2020-07-19  (Preprint being edited for publication.)
https://www.biorxiv.org/content/10.1101/2020.07.18.210161v1

uses autocrine in the title, but refers to the vitamin D based autocrine signaling as also potentially involving paracrine signaling.  The molecular mechanisms all involve VDR in the cell, which according to the 1991 Labrie diagram, is intracrine signaling.  There is no mention of intracrine.

I am having enough trouble as it is getting doctors,  immunologists etc. to understand vitamin D's role in the immune system without these terminological complications.  Two recent immunology textbooks Janeways 9th 2017 and Abbas 10th 2021 total 1500 pages and do not mention vitamin D in their indexes.  The only mention of autocrine and paracrine signaling is in Janeways 9th, regarding cytokines in which autocrine is defined as affecting the behaviour of the cells which release the cytokine, without reference to the location of the receptor. 

For now, to the possible annoyance of Professor Hewison, I am sticking with autocrine signaling, in accordance with McGregor et al. who provide explicit details of the process, with this note about intracrine being regarded by some people as a more appropriate term.

Another reason for my choice is the highly cited 2009 article, cited below #extra-renal, of which Professor Hewison is the lead author, which mentions only autocrine and paracrine signaling.

This knowledge of vitamin D autocrine signaling has been developed since the mid-2000s.  As far as I know, there are no accurate estimates of how many types of cell use vitamin D autocrine or autocrine-paracrine signaling.   All types of immune cell use this as you can read in the Charoenngam & Holick article linked to below.

For more in-depth material, a good place to start might be articles which cite a 2010 article,  Autocrine and Paracrine Actions of Vitamin D by  Howard A Morris and Paul H Anderson. Also: Vitamin D metabolism and signaling in the immune system (2012), Vitamin D and immune function: autocrine, paracrine or endocrine (2013)  and Vitamin D and immune function (2013).  These processes were not always described as "autocrine" or "paracrine", but these are the proper terms to use now.

As far as I know, there is no research article which presents autocrine (or intracrine) signaling and/or paracrine signaling in an easy to understand manner.  So I created this web page.


Here are two other terminological and conceptual failings frequently found in vitamin D research articles:

#01-compounds

D3, 25OHD and 1,25OHD - the three main vitamin D compounds




D3 cholecalciferol. [WP]   This is produced by 295 to 297 nanometre wavelength UV-B light acting on 7-dehydrocholesterol in the skin.  It can also be ingested in food or supplements.  While this plain D3 directly protects the endothelial cells which line our blood vessels [Gibson et al. 2015], all its other currently known roles in the body rely on it being converted in the liver (there may also be some conversion in cells out side the liver), over a period of days to a week, by the enzyme vitamin D 25-hydroxylase (encoded by the CYP2R1 gene, a name sometimes given to the enzyme itself) to 25OHD.  (Another enzyme encoded by the CYP27A1 gene does the same thing and so produces some of the 25OHD.)

The numbers indicate carbon positions.  Most hydrogen atoms are not shown.  The special trick to producing this from 7-dehydrocholesterol is to use 295 to 297 nanometre UV-B light to break a ring between carbon 9 and 10. 



25OHD calcifediol = 25 hydroxyvitaminD3 = calcidiol [WP].  This has an OH oxygen-hydrogen hydroxyl group at the 25 position, in place of the H (not shown) which was there.  25OHD circulates in the blood, mainly bound to the vitamin D carrier protein and albumin.  It is also absorbed into fatty tissue, as is D3.  Vitamin D blood tests measure the total bound and unbound level of 25OHD.  This level is the most important part of the whole vitamin D system, however, depending  the amount which is unbound to the vitamin D carrier protein and perhaps the albumin proteins may affect the amount of 25OHD which diffuses from the bloodstream into the interstitial fluid between the cells, and through their cell membranes into the cytosol of the cells. .

Neither D3 nor 25OHD bind strongly to the vitamin D receptor [W] which is a complex protein far bigger than these molecules.




1,25OHD calcitriol [WP] = 1,25 dihydroxy vitamin D = 1,25(OH)2 vitamin D, is produced by a second enzyme, 1-hydroxylase, encoded by the CYP27B1 gene, which adds an OH hydroxyl group at the 1 position to 25OHD.   This happens in the kidneys and inside many types of cells, including immune cells.

1,25OHD binds strongly to, and so activates, the vitamin D receptor.

There is another enzyme CYP24A1 which can add an OH hydroxyl group to the 24 position of 25OHD and 1,25OHD, which is an irreversible process.  The resulting molecules are degraded and excreted.  The activity of this enzyme scales up with increasing circulating 25OHD levels, and so gives rise to a strong self-limiting process which reduces high 25OHD levels.  This accounts for the curves in the 25OHD by bodyweight and D3 intake graph from Ekwaru et al. 2014 at 01-supp/a-ratios/ .  This self-regulation makes it very hard to attain potentially toxic 25OHD levels.  Above 150ng/ml (375nmol/L) there is a risk of hypercalcemia [WP].


#02-nothorm

Autocrine (including intracrine) and paracrine signaling with 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D and differs from 1,25-dihydroxyvitamin D as an endocrine signaling agent (hormone) and is not significantly affected by this very low, stable, level of circulating (hormonal) 1,25-dihyroxyvitamin D

Vitamin D based autocrine signaling (potentially AKA intracrine signaling) and paracrine signaling, at least in immune cells, is not a continual process.  The signaling system is activated in a particular cell when certain conditions are detected.  This causes both VDR (vitamin D receptor) and the 1-hydroxylase enzyme to be produced in the cytosol, whereupon the enzyme converts the 25OHD which should already be there (and be supplied from outside the cell, as it is consumed) into 1,25OHD, which immediately binds to a VDR molecule.  The bound complexes find their way to the nucleus where they alter gene transcription, typically upregulating and downregulating the transcription of dozens of genes. 

The exact details of what activates the signaling process (the upregulation of transcription of VDR and 1-hydroxylase enzyme genes, which leads to their synthesis in the cytosol) varies from one cell type to the next.   Likewise, the exact details of which genes are upregulated and downregulated when the VDR-1,25OHD bound complexes enter the nucleus, varies from one cell type to the next.

Vitamin D based autocrine/paracrine signaling is the same general system in multiple cell types.  Tens or hundreds of millions of years of evolution have used these systems for a variety of purposes - a different purpose in each type of cell - with the activating conditions and the changed cell behaviour being entirely cell-type specific.  So there is no generalised way of describing these signaling systems in terms of what activates them and what changes they create in cell behaviour - since these vary widely from one cell type to the next.

In this section I explore something I haven not seen explicitly tackled in the research literature.  Can anyone can point me to where this has been tackled?

The question is "To what extent, if any, are autocrine signaling operations in immune cells affected by the relatively stable, and very low, level of hormonal 1,25OHD in the bloodstream?"  As far as I can see, this hormonal level is too low to significantly affect autocrine signaling, so moderate changes in that hormonal level would also have no significant effect.

A related question is the same regarding paracrine signaling.  I don't know of any measurements of the levels of 1,25OHD diffusing to nearby cells, but these levels will be somewhat or perhaps a lot lower than the levels at which 1,25OHD is generated intracellularly.  For paracrine signaling to work at all, it needs to be sensitive to these diffused levels, and not significantly affected by moderate changes in hormonal 1,25OHD which presumably diffuse from the bloodstream into the interstitial fluid between the cells.    I can't answer this question quantitatively, but it seems likely that paracrine signaling involves diffused levels of 1,25OHD which are well above the hormonal level of 1,25OHD.

Here is a description of the one hormonal function of the vitamin D compounds.  A hormone is a compound in blood circulation, whose level (concentration) is controlled, with that level affecting the behaviour of one or more cell types.

Carefully regulated, very low, levels of 1,25OHD are produced in the kidney from 25OHD and are put into circulation in the blood as a hormone to regulate calcium-bone metabolism.  This hormonal 1,25OHD has a half life of around 6 hours, which is much shorter than the month or so half life of 25OHD (or shorter with higher concentrations and longer if the levels are very low, such as below 20ng/ml) . 

In one study, 25OHD levels averaged 36ng/ml (91nmol/L 36 parts per billion by mass), which is quite a good level, and around twice what people attain without much high elevation direct sunlight skin exposure or proper vitamin D supplements. (There is little D3 in food or multivitamins, and the UK's 0.01mg 400IU a day is a scandalously small amount.)  The kidneys convert enough circulating 25OHD into 1,25OHD to maintain whatever level of circulating (hormonal) 1,25OHD is needed throughout the body to maintain proper calcium levels in the blood, which are sensed by the parathyroid glands to the control parathyroid hormone level, which controls the kidneys' conversion rate.  As long as 25OHD levels are above about 20ng/ml, the kidneys will have no trouble converting it fast enough to maintain the desired circulating 1,25OHD level.

In this study, the average circulating (hormonal) 1,25OHD level was 0.045ng/ml (45 parts per trillion, 0.111nmol/L, which is 1/800th of this 36ng/ml 25OHD level.  So every 6 hours the kidneys convert about 1/1600th of the circulating 25OHD into circulating 1,25OHD.  Over a month, this requires about 1/12th of the circulating 25OHD.  Since the half life of this circulating 25OHD is a month or so, we can guesstimate that about 1/6th of 25OHD lost every month is due to conversion in the kidneys to hormonal 1,25OHD.  The other 5/6th of the loss must be due to its use in autocrine (AKA intracrine) (and sometimes paracrine) signaling in many cell types all over the body, and to the 25OHD being degraded by the 24-hydroxylase enzyme.

If 25OHD levels were very low, such as 10ng/ml or 20ng/ml (which is disastrously common all around the world), then the kidneys would generally maintain their 1,25OHD conversion to maintain the level required for proper calcium-bone metabolism, but the autocrine/paracrine signaling systems of numerous cell types, including all type of immune cell, would not be working properly, and so would consume less 25OHD per month, with very little being degraded by the 24-hydroxylase enzyme.

However, this would not work quite so well and adults would be at risk of osteoporosis.  25OHD levels of 10ng/ml or less in children causes rickets [WP] - failure of the bones to grow strong and straight.

So at healthy levels such as 40ng/ml to 80ng/ml, we can assume that (very approximately) only a tenth or less of the 25OHD produced from D3 is used by the kidneys for the one hormonal function of the vitamin D compounds.

While 1,25OHD (discovered in 1972) is the best known has a hormone for its role circulating in the blood, its production (in the kidneys, as just described) is not where most of the D3 (converted to) 25OHD vitamin D is used.  Kidney conversion to hormonal 1,25OHD was the only known use until about 1979 when extra-renal (outside the kidneys) conversion to 1,25OHD was first discovered (Gray et al. 1979).  In 2007 an important article was published, discussing vitamin D autocrine and paracrine signaling:

Extra-renal 25-hydroxyvitamin D3-1alpha-hydroxylase in human health and disease
Martin Hewison et al. J. Steroid Biochem. Mol. Biol. 2007-03
https://www.sciencedirect.com/science/article/abs/pii/S0960076006003864 (Paywalled.)
https://sci-hub.se/10.1016/j.jsbmb.2006.12.078
408 Google citations.

These researchers used some macrophages and monocyte derived dendritic cells, both with their autocrine/paracrine signaling systems turned on, to find out how their conversion of 25OHD to 1.25OHD was affected by differing levels of 25OHD: 2, 20 and 60ng/ml.  The levels of 1,25OHD produced, after 48 hours, were (Fig 1 levels divided by 2.5 to give ng/ml) approximately 0.013, 0.12 and 1ng/ml.

The 0.12ng/ml 1,25OHD (resulting from 20ng/ml 25OHD supply to the cells) only marginally affected the gene transcription and protein synthesis which autocrine signaling in the macrophages drives.  This is upregulation of CD14 [WP] and downregulation of three other proteins (Fig. 2).  The 1ng/ml 1,25OHD level, produced when 60ng/ml 25OHD was supplied to the macrophages) fully upregulated CD4 and downregulated the other three proteins - the effect was just as strong as when 40ng/ml 1,25OHD was added to the cells.

Some important points arise from the abovementioned research:

#03-minlev

At least 50ng/ml 25OHD blood levels required for good immune system function

The importance of proper (at least 50ng/ml = 125nmol/L = 1 part in 20 million by mass) levels of 25OHD is not widely enough known.   While lower values may be sufficient for the kidneys to maintain the proper level of hormonal 1,25OHD, we need at least this level of 25OHD for numerous types of cell - especially immune cells - to function correctly. 

The target range of 40 to 60ng/ml (100 to 150nmol/L) was stated in 2008 by 48 leading researchers and MDs in the Call to D*Action: https://www.grassrootshealth.net/project/our-scientists/   This approximately 50ng/ml level was fully justified by the research of Quraishi et al. 2014, mentioned in a section below: #04-quraishi .

This 2020 review article, co-authored by the world's leading vitamin D researcher, (Prof. Michael Holick) also calls for 40 to 60ng/ml 25-hydroxyvitamin D:

Immunologic Effects of Vitamin D on Human Health and Disease
Nipith Charoenngam, Michael F. Holick 2020-07-15
Nutrients 2020, 12(7), 2097
https://doi.org/10.3390/nu12072097

This article and another one:

Disassociation of Vitamin D’s Calcemic Activity and Non-calcemic Genomic Activity and Individual Responsiveness: A Randomized Controlled Double-Blind Clinical Trial
Arash Shirvani, Tyler Arek Kalajian, Anjeli Song & Michael F. Holick, Nature Scientific Reports 2019-11-27
https://www.nature.com/articles/s41598-019-53864-1

report on hundreds of genes which are upregulated or downregulated by vitamin D in a sample of white blood cells.  Below, I explain how the upregulation occurs, but not the downregulation since I don't yet understand the molecular mechanisms. All these genes are affected as part of autocrine/paracrine signaling in an unknown number of cell types, including all immune cell types. 

So there seems to be a large and so-far undefined number (I guess dozens to hundreds) of cell types who respond to their circumstances in part, at least, via vitamin D based autocrine/paracrine signaling.

This means vitamin D (the three compounds in general, but in the cells themselves, just 25OHD and 1,25OHD) are extraordinarily important for most or all systems of the body.  The scope of vitamin D's role in the body extends beyond the proteins for which these specific genes provide the instructions, because some of these genes involve proteins which affect histones [WP].   Histones are proteins which physically organise the long DNA molecules of the chromosomes, 1.8 metres in total.  An important role of the histones is to unwind particular regions of the DNA so its genes can be copied into messenger RNA molecules and so direct the cell's protein making machinery.  To whatever extent vitamin D autocrine/paracrine signaling affects histones, it therefore affects numerous other aspects of the cell's ability to perform its functions. 

40 to 60ng/ml (100 to 150nmol/L) was also suggested as the proper target range in this 2019 article (24 citations):

Daily oral dosing of vitamin D3 using 5000 TO 50,000 international units a day in long-term hospitalized patients: Insights from a seven year experience
Patrick J.McCullough, Douglas S.Lehrer, Jeffrey Amend
Journal of Steroid Biochemistry and Molecular Biology V189, May 2019

https://www.sciencedirect.com/science/article/abs/pii/S0960076018306228 (Paywalled.)
https://sci-hub.se/10.1016/j.jsbmb.2018.12.010

This article also discusses the benefits some people find from much higher 25OHD levels, for suppressing inflammatory disorders such as psoriasis and rheumatoid arthritis.  Please see https://vitamindstopscovid.info/06-adv/ for more on this and how it relates to our lack of helminths (intestinal worms).

Please also see the recent article from MDs in Dubai who had great success with COVID-19 patients by either previously raising their 25OHD levels to the 40 to 90ng/ml 100 to 225nmol/L levels or by using the same bolus D3 and then body-weight ratio continuing supplemental D3 intakes on newly diagnosed hospitalised COVID-19 patients.  The link and my summary is at: https://aminotheory.com/cv19/#2020-Afshar .

Here is another recent research article:

Editorial – Vitamin D status: a key modulator of innate immunity and natural defense from acute viral respiratory infections
A. Fabbri, M. Infante, C. Ricordi Eur Rev Med Pharmacol Sci 2020; 24 (7): 4048-4052 2020-04-05
https://www.europeanreview.org/article/20876

They mention that 40 to 60ng/ml circulating 25OHD is required for the autocrine signaling system of immune cells to function properly

The text (in the quote below) "the beginning point of the plateau where the synthesis of the active form calcitriol becomes substrate-independent" requires some explanation for non-specialists.  The 1-hydroxylase [WP] enzyme  is a large, complex protein, whose actions are powered by some other molecules which are changed in the process.  The authors are discussing the hydroxylation of 25OHD to 1,25OHD, which is a crucial early step in autocrine signaling.  There are multiple 1-hydroxylase enzyme molecules in the cell, and each converts one 25OHD molecule at a time to 1,25OHD.  The speed of this conversion is important, since if it is too slow, then the 1,25OHD levels in the cytosol (main body of the cell, where this happens - not in the nucleus) will not raise to a high enough concentration (as noted above, around 1ng/ml (1 part per billion by mass) that a sufficient number of these 1,25OHD molecules will bind with vitamin D receptor molecules, after which some of these bound complexes migrate (or at least diffuse) to the nucleus, as I will describe properly below.

There are a few 24-hydroxylase enzyme molecules in the cell, converting any 1,25OHD they find to inactive 1,24,25OHD which is broken down into compounds which are excreted.  (This enzyme does the same thing to the more numerous 25OHD molecules in the cell: convert them to 24,25OHD which is broken down and taken away.)  This serves two purposes.  Firstly, mopping up any hormonal (from the bloodstream) 1,25OHD which diffused into the cell, to reduce the degree to which it might activate the rest of the autocrine signaling system.  Secondly, to slowly mop up 1,25OHD previously produced by the autocrine signaling system operating normally, so that the levels drop after this system is no longer activated.   In a further twist, some of these enzyme molecules are formed differently and don't convert 25OHD or 1,25OHD, they just bind to them for a while and so are described as decoys. [Cantorna et al. 2015, and also Hewison et al. 2007, above.]

If there is no 25OHD, obviously the autocrine signaling system cannot work.  If there is too little, then it will work too slowly, or not work properly - so the cell will not respond fully to its new circumstances and our health will suffer.

The enzyme itself is not changed - it is a catalyst.  When, by random thermal motion, a molecule of 25OHD is in the right position in the enzyme's active site, the enzyme replaces the H in the 1 position with an OH hydroxyl group, at which time the newly-formed 1,25OHD is no longer so attracted to the enzyme's active site, and floats away.  

The 25OHD molecule, up to the point where it is converted to 1,25OHD, is the substrate.  The authors imagine a graph with 25OHD concentration being the horizontal axis and the total rate of conversion to 1,25OHD being the vertical.  The plateau they refer to is where the rate of conversion no longer rises linearly (upwards and to the right) with 25OHD concentration, due to the limiting factor being mainly the enzyme's own intrinsic speed of conversion, when it has it hardly as to wait for a fresh 25OHD molecule to arrive in its active site.  

We also believe that maintenance of circulating 25-hydroxyvitamin D levels of 40 - 60ng/ml would be optimal, since it has been suggested that concentrations amounting to 40ng/ml represent the beginning point of the plateau where the synthesis of the active form calcitriol becomes substrate-independent [2011-Hollis err] [2018-Wagner].

Additionally, serum 25-hydroxyvitamin D levels of approximately greater than or equal to 40ng/ml could provide protection against acute viral respiratory infections, as demonstrated in a prospective cohort study published in PLoS One and conducted on 198 healthy adults [2020-Sabetta].  To reach these concentrations in adults, a dietary and/or supplemental intake of vitamin D up to 6000 IU/day – deemed to be safe – is required.  However, elderly subjects, overweight/obese and diabetic patients, patients with malabsorption syndromes, and patients on medications affecting vitamin D metabolism may require even higher doses under medical supervision.


The authors mean that if 25OHD levels (in the blood) are around 40ng/ml or more, then this leads, via diffusion - there being no active transport of 25OHD from the bloodstream into the fluid between the cells and across the cell's membrane - to a concentration of 25OHD in the cell to start with which enables the enzyme to work at close to its full speed converting these 25OHD molecules to 1,25OHD.   Also, this 25OHD level in the blood is required to maintain the 25OHD levels in the cell as some of the 25OHD is consumed by the conversion process, so the enzyme is not slowed down by having to wait for a fresh 25OHD molecule to arrive in its active site. 

The key thing to remember is that 25OHD levels are very low.  A healthy level is 50ng/ml, but many people, without supplements, never achieve this. So for many people, average levels are 1/2 or even as low as 1/10th of this.  50ng/ml (50 parts per billion) is only one part by mass of 25OHD per 20,000,000 parts by mass of all the water and other compounds in the cell.  So these are quite rare molecules.   A 70kg person only needs a gram of D3 every 22 years, about 1/3  to 1/4 of which is converted to 25OHD in the liver, to maintain this healthy level.  (50 parts per billion is like a 3.7mm cube of water in a cubic metre of water.)


You probably began reading this page thinking of the COVID-19 crisis, the influenza crisis and perhaps the sepsis crisis.  Now you are contemplating lonely 25OHD molecules being jostled around by the thermal vibrations of surrounding molecules (mainly water) until one of these molecules:



This is adapted from https://commons.wikimedia.org/wiki/File:Calcifediol_3D_ball.png with numbering from Boudal-Attar-D3.png from Vitamin D and Autoimmune Disease, Ayah Boudal and Suzan Attar, (book chapter from Insights into Rheumatology 2012 Researchgate).  The molecule is oriented differently from its orientation in the  diagrams at the start of this page.  Here is a higher resolution image, with original colouring and all carbon numbers: Calcifediol-3D-ball-numbers.png
  1. Arrives very close to the active site of the much larger enzyme molecule.  This is 3 dimensions of movement over large distances (one such molecule on average per ~320 nanometres cubed) compared to the size of the 25OHD molecule (~0.2 nanometers) and the enzyme molecule:



    Adapted from paywalled article https://www.degruyter.com/document/doi/10.1515/jpem-2013-0183/ Sci-Hub: https://sci-hub.se/10.1515/jpem-2013-0183 Wei-Wei Hu et al. A novel compound mutation of CYP27B1 in a Chinese family with vitamin D-dependent rickets type 1A 2013-11-07 .

  2. Is pointing in exactly the right direction for it to fit.  This needs to be correct in 3  rotational dimensions in order to align the end-to-end axis of the molecule with the axis of its  position in the enzyme's binding site.

  3. Is rotated correctly along its axis - this is 1 axial rotational dimension - so the 25OHD molecule is precisely aligned with the matching outer electron orbitals of the atoms of the enzyme's binding site..
When this happens, the positive and negative charges (due to negatively charged electron orbitals being off-set from the positively charged nucleus they surround) on particular parts of the two molecules will draw them closer, the 25OHD will be fully docked, and the enzyme and its co-factor molecules will do their work of attaching the OH to the 1 position group.

This probably seems a long way from COVID-19, but it is absolutely germane If everyone in the world had 40ng/ml or ideally 60ng/ml more 25OHD in their blood, then:
  1. The enzymes in all their cell types which use vitamin D for autocrine/paracrine signaling would not be waiting long for another 25OHD molecule to dock.

  2. So they would produce 1,25OHD at a perfectly healthy rate whenever the autocrine signaling system is activated.

  3. The autocrine signaling systems of all cell types (including all types of immune cell) would work correctly, making then respond fully and rapidly to their changing circumstances.

  4. Although there are numerous other factors affecting total immune system performance, this would mean that the current vitamin D deficiency epidemic would not exist - and it is low vitamin D which is the primary cause of some immune responses being weak, while others are dysregulated - meaning overly-aggressive, hyper-inflammatory and self-destructive.   These weak and dysregulated immune responses are the primary or sole reason why some people who are infected with SARS-CoV-2 develop severe COVID-19.

  5. So almost all people would fight off the SARS-CoV-2 infection without serious symptoms.  Likewise flu.  Also, very few people would develop sepsis, Kawasaki disease or Multisystem Inflammatory Syndrome.

    (Note: there is a lot of interest in the idea that high vitamin D levels will substantially reduce the chance of being infected with COVID-19 for any given vital insult.  I see no evidence that this is more than a marginal effect.  The most important point, for all society, is the next one, followed by the just-mentioned great reduction in average severity.)

  6. For those infected, average total quantities of viral shedding would also be greatly reduced, so fewer people would become infected.  COVID-19 would not spread very much at any time of year.  Likewise flu.

  7. So there would be no COVID-19 crisis, with no need for lockdowns, social distancing, vaccines or masks.  The few who did become seriously ill could be treated with oral 25OHD (calcifediol) and D3 ../04-calcifediol/  as well as other early treatment techniques.  (I plan to add a proper page on these techniques.)

You now have an understanding of a crucial part of the current global crisis down to a molecular level - and if you want to, you can look up the gory details of the virus, the ACE2 receptor, the destruction of the endothelium, the hypercoagulative state of the blood and the microembolisms and larger clots in the lungs, brain, heart, spinal cord, liver kidney etc.

None of those gory details would matter, because they would not exist, if everyone had enough vitamin D.   70kg adults, on average, to attain about 50ng/ml (125nmol/L) 25OHD, without relying on UVB skin exposure, or the small amounts of D3 in food, need to ingest 45 milligrams of D3 year = a gram every 22 years.  This is 0.125mg 5000 IU a day.   Pharma grade D3 costs about USD$2.50 a gram, ex-factory. 

Please see https://vitamindstopscovid.info/01-supp/ for D3 supplemental intakes, as ratios of bodyweight, which I derived from the work of Ekwaru et al. 2014.


#04-quraishi

25OHD requirements for immune cell autocrine/paracrine signaling as indicated by hospital infection rates following surgery

Here is another way of understanding the need for proper 25OHD levels around or above 50ng/ml (125nmol/L).  The following graph comes from research into the risk of infections in people (all obese) who had just been operated on for Roux-en-Y gastric bypass [WP]. 

This is a weight-loss surgery with numerous problems due to malabsorption of fats, iron, and other nutrients including vitamin D3 and due to overly rapid, uncontrolled, absorption of carbohydrates.  It is a highly regarded operation in the USA - I have not heard of anyone in Australia performing it.  However, less drastic operations such as gastric banding are gaining favour over Roux-en-Y.  All those who underwent this operation and who were subjects in the Quraishi et al. retrospective analysis had this operation to treat morbid obesity.  This seems crazy to me when they should first try to reduce the imbalances which drive their obesity: robust supplements for all micronutrients including especially vitamin D3, no fructose, no caffeine and so less need for alcohol, nicotine and anti-depressants / anxiolytics.  However, morbid obesity is a deadly medical problem and is very difficult to tackle - hence the attraction of these drastic surgical interventions.

Association Between Preoperative 25-Hydroxyvitamin D Level and Hospital-Acquired Infections Following Roux-en-Y Gastric Bypass Surgery
Sadeq A. Quraishi et al. JAMA Surg. 2014-02 https://jamanetwork.com/journals/jamasurgery/fullarticle/1782085


This PNG is from my Inkscape version combining two similar graphs, made from the vectors in the PDF.  So the red and purple lines, and the scales, are direct from the article's graphs, not a result of me trying to copy them by some approximate method.  This is a version of the graph  made in September 2020, for an immunologist here in Victoria, Australia.



Please think of these graphs whenever you read of individual and average 25OHD levels in people who are not adequately supplementing D3 and who do not get very substantial UVB skin exposure (which damages DNA and which I do not recommend).  Their levels are typically between 5 and 20 or 25ng/ml.

The graphs depict how the risk of infections in hospital - either directly resulting from the surgery or due to other reasons - vary with vitamin D 25OHD levels, for 770 patients.

Low rates of infections occur when the immune system's innate [WP] and adaptive ([WP] antibodies etc. ) responses are functioning properly.  The failures are due to weak immune responses which directly combat the (primarily bacterial) pathogens which cause these infections, as in the first autocrine signaling example below.  (The second McGregor et al. example below concerns innate immune system regulatory lymphocytes which, when their autocrine signaling fails due to lack of 25OHD, cause trouble by producing pro-inflammatory cytokines for longer periods than they should.  This failure causes other immune cells to destroy healthy cells, especially in the blood vessels of the lungs.  This causes or at least strongly drives severe COVID-19, but is not likely to be important in the infections in hospital which are the subject of Quraishi et al.'s research.)

With one potential exception, wherever the graphs rise above about 0.025, this is due to autocrine signaling not working properly in some - probably many - types of immune cell.  The potential exception is that that the higher D3 levels which give rise to the higher 25OHD levels are also directly useful (without involving autocrine signaling) in the protection of endothelial cells [Gibson et al. 2015].  I have not been able to quantify how important this is, and I suspect the main cause of these infections is the failure of the innate immune system to rapidly defeat bacteria.

The raised risks of infection indicate dysfunction of autocrine/paracrine signaling due to inadequate 25OHD.   Eyeballing this we see that the 40ng/ml minimum recommendations mentioned above don't go quite far enough.  The evidence of this substantial research (770 subjects, in one hospital, with all the researchers being from Harvard Medical School) indicates that we should be aiming for at least 55ng/ml, at least in these obese adults.  There is some scatter in the measurement of 25OHD levels.  For this reason, and to simplify things a little, I write of this research as if it suggests that at last 50ng/ml is required for proper immune system function.

This research does not tell us directly what 25OHD levels are required for Th1 regulatory lymphocytes to avoid the pattern of being stuck in their pro-inflammatory startup program indefinitely, as described in McGregor et al. below.  There are surely other regulatory immune cells which behave in a similar fashion - weakening or pathologically over-strengthening inflammatory responses if they don't have enough 25OHD.

However, it is reasonable to guess that they too would generally work reasonably well only when 25OHD was also at or above 50ng/ml .  The exact 25OHD levels required to suppress hyper-inflammatory immune responses surely vary considerably from one person the next, according to individual genetic variation, in a context in which many people have a problem with these responses, by way of auto-immune inflammatory conditions, due to no longer having helminths.  Please see https://vitamindstopscovid.info/06-adv/ for more information on this.   I expect that if we all had helminth infections, we would generally not need such high 25OHD levels to suppress these autoimmune problems.   I will add to this page a link to recent research from Ethiopia which indicates that active helminth infections reduce the risk of severe COVID-19 by 75%!

Helminths are known to suppress inflammatory responses, which are primarily directed at helminths and other multicellular parasites.  They are not known to substantially suppress the innate and adaptive responses to viruses, fungi or bacteria - and it is primarily these anti-bacterial innate and adaptive responses which are directly weakened by the lower than 50ng/ml 25OHD levels which are indirectly measured in Quraishi et al. by way of their weakening increasing the risk of infection.


#05-auto
The following description is mainly of autocrine signaling, using vitamin D (25OHD being converted to 1,25OHD).  Paracrine signaling is easy to understand as an extension of this.

Step 1 - producing vitamin D receptor and 1-hydroxylase enzyme molecules

The diagrams below are adapted from a diagram in this 2011 article which has a good description of vitamin D autocrine signaling in a particular type of immune cell, although the term autocrine is not used:

Antibacterial effects of vitamin D
Martin Hewison,  Nature Reviews Endocrinology v7 2011-01-25
https://www.nature.com/articles/nrendo.2010.226 (Paywalled.)
https://sci-hub.se/10.1038/nrendo.2010.226 
339 Google citations.

Here is a description of autocrine (or intracrine, as mentioned in #00-term above) signaling which assumes an interest in cell biology, but little prior knowledge.

In this example from Prof. Martin Hewison, we learn how toll-like receptors [WP] on the cell membrane of some types of monocytes [WP] - in this case a macrophage [WP] respond to bacterial infections.  The same principles of vitamin D autocrine signaling apply to other types of cell, including the Th1 regulatory lymphocytes discussed below (McGregor et al.), although the stimulus for activating autocrine signaling is totally different to the bacterial fragments in the current example, and the response of the lymphocyte is also entirely different.



In this cell type, fragments of pathogens activate toll-like receptors which are embedded [WP] in the cell membrane, and which change their shape so the part of the molecule inside the cell (in the cytosol [WP]) causes some other signaling molecules to migrate to the nucleus and upregulate the transcription of two genes: for the 1-hydroxylase enzyme and for the vitamin D receptor (VDR) protein.

Those signaling molecules are cell-type specific, and somehow cause transcription enzymes to make mRNA (messenger RNA [WP]) copies of the information in those genes.  These multiple mRNAs migrate out of the nucleus, to the cytosol, where they are found by ribosomes [WP] which work along each mRNA molecule, following its instructions of which amino acids to assemble into the protein chain.  This is called translation

When each ribosome reaches the other end of the mRNA molecule, it has produced one chain, which folds of its own accord to become a complete single molecule of protein.  This creates some number (I guess hundreds or thousands) of complete, operational, 1-hydroxylase enzyme molecules and likewise vitamin D receptor molecules.

Step 2 - converting 25OHD to 1,25OHD molecules and these binding to vitamin D receptor molecules

25OHD is carried in the blood plasma primarily bound to vitamin D binding proteins [WP], with a lower proportion bound less strongly to albumin [WP] proteins.  A small proportion of these 25OHD molecules (red discs) are free to diffuse from the plasma, into the interstitial fluid between cells (in the case of cells which are not in the bloodstream or in the walls of blood vessels) and then they  diffuse across the cells' lipid bilayer [WP] plasma membrane into the cytosol of the cell.  

D3 has only one hydroxyl group, in the 3 position with all its other sides made up of hydrogen atoms.  So it is soluble in oils but not much in water.  25OHD has two hydroxyl groups and so is more soluble in water.



Once the newly created 1-hydroxylase enzymes start appearing in the cytosol, assuming there is an adequate concentration of 25OHD molecules (red discs) - which there will be if blood levels are 50ng/ml or more - then it doesn't take long for one of these 25OHD molecules to find its way to the active site of the enzyme molecules, be hydroxylated at the 1 position, and be ejected back into the cytosol as 1,25OHD molecules, (green discs).

By now there will be some number of vitamin D receptor VDR molecules and the freshly made 1,25OHD molecules find their way (as described above, with random thermal motions and rotations) into the active site of one of these receptors, where the two are strongly attracted and stick together as an activated receptor complex.

This newly produced 1,25OHD is functioning as an autocrine agent which binds to these vitamin D receptor molecules.

This step is identical for the vitamin D based autocrine signaling systems of all cell types.

Step 3 - Activated receptor complexes diffuse or migrate to the nucleus where they alter gene transcription and so protein translation

When a 1,25OHD molecule binds to the receptor molecule, this changes the shape of the receptor molecule and causes some of them to migrate into the nucleus.  (I guess only a subset of them migrate to the nucleus, so perhaps is is diffusion, rather than them all marching off in the direction of the nucleus.)  Articles mention them "translocating" to the nucleus, but this just means "move " and I know of no active transport or guidance system for them doing this, so for now I assume that the activated 1,25OHD-VDR complexes simply move around at random, due to thermal motion, and that some subset of them diffuse into the nucleus.

There the activated receptor complexes find their way (by diffusion, I guess) to another molecule (retinoid X receptor [WP], which is related to vitamin A and is not shown in these diagrams) with binds to them as well and the entire heterodimer [WP] complex then finds its way to particular patterns of DNA which are exposed (according to how the DNA of the various chromosomes are wrapped around and otherwise organised by histones [WP]) and ready to accept them.  These are the VDRE (Vitamin D Response Elements [WP]) and they are upstream of a particular gene which this process is intended to increase or decrease the copying of.   (The whole human genome has thousands of such genes, with a VDRE upstream.  By various means, each cell type exposes only these to being bound by the heterodimer complex - the particular genes which this cell needs to be copied in order to respond to its circumstances properly.)



Once the VDRE section of DNA has the heterodimer attached, in some circumstances this signals DNA copying enzymes to start work there, copying the data in the downstream gene into messenger RNA molecules.  In others, it reduces the amount of copying of this gene.  (Can anyone point me to a good description of these mechanisms?)

In principle, if this process of activated receptor complexes finding their way to these transcription regulator molecules was highly guided, then there would only need to be a handful of 25OHD molecules converted to 1,25OHD, perhaps by a single or a few enzyme molecules, and likewise there would only need to be a handful of vitamin D receptor molecules.

However, since the processes are unguided (diffusion) or at least not very efficient, and since the activated complexes and probably the 1,25OHD molecules would have relatively short half-lives (of their own accord, or by enzymes breaking them down, to get rid of them once the conditions which activated autocrine signaling no longer occurred) then there needs to be quite a quantity of both 1,25OHD and vitamin D receptor molecules ready to bind together.  This requires continual conversion of 25OHD to 1,25OHD, since the 1,25OHD molecules have relatively short half-lives.  As noted above, to fully alter the gene translation process to change the cell's behavior, it seems there needs to be around 1ng/ml (1 part per billion by mass) 1,25OHD in the cytosol of the cell.

Considering that:
With upregulated gene copying, the newly copied mRNA molecules leave the nucleus and go into the cytosol, where ribosomes run along them, making the proteins they contain the instructions for.  (For downregulation, fewer of these mRNA molecules are produced than previously.)  These proteins are the ones which make the cell respond to its changed circumstances.   In some cells, these may be exported to kill pathogens, or to kill infected cells.  In others, the proteins may cause the release of pro-inflammatory or anti-inflammatory cytokines [WP] - signaling molecules which control activities of other types of immune cell which are nearby.

The alterations to gene transcription alter the mix of mRNAs in the cytosol and so (translation) the quantities of proteins produced by the ribosomes which run along them.  (mRNAs have quite short lives, so for continual protein production a continual supply of them via transcription is required.)

This altered set of protein products is what drives the cell to alter its behaviour.  In this example, the altered behaviour sets the cell up for engulfing and digesting bacteria.

In the McGregor et al. example below, when the autocrine signaling systems of a Th1 lymphocyte is activated and works properly, the lymphocyte switches to its shutdown program in which it produces less of a pro-inflammatory cytokine and more of a anti-inflammatory cytokine.


Paracrine signaling

One part of paracrine signaling is depicted at the bottom left of the above diagrams: some of the newly produced 1,25OHD diffuses out of the cell and reaches nearby cells. 

As noted above #02-nothorm, the concentration of this 1,25OHD is probably around 1ng/ml (1 part per billion by mass) when the autocrine signal is is fully activated and there is sufficient 25OHD to achieve this.  This 1ng/ml is much higher than the very low levels of 1,25OHD present in the bloodstream as a hormone to regulate calcium-bone metabolism, around 0.045ng/ml (46 parts per trillion).

The newly produced 1,25OHD is functioning as a paracrine agent when it diffuses out of the cell, and makes its way to other nearby cells where - by one means or another - this increased local level of 1,25OHD is detected in a way which alters the behaviour of those nearby cells.  As far as I know, VDR is only found in the cytosol and nucleus of the cell - it is not located in the cell membrane, ready to detect 1,25OHD outside the cell.  So, as far as I know, paracrine signaling works by extracellular 1,25OHD, diffused from where it was produced in nearby cells, diffusing into the cytosol and binding to VDR molecules there.  Then, some of the bound complexes diffuse into the nucleus and alter cell behavior as just described for autocrine signaling.


The McGregor et al. article on autocrine signaling failing in Th1 lymphocytes due to lack of 25OHD

If most or all of the above makes sense to you, then you are in a good position to either read the entire McGregor article, or at least my summary and discussion of it, at:

https://aminotheory.com/cv19/icu/#2020-McGregor

The basic summary is below.  The above page has a more extensive summary and discussion.

Then, you will have a real, cellular and molecular level understanding of some of the most important reasons why the world is going to hell in a handbasket at present, with the twin crises of COVID-19 and of the attempts to protect people from this disease by lockdowns etc.

It would be much easier if everyone took vitamin D supplements to raise their 25OHD levels to the ancestral levels which enable our vitamin D autocrine signaling systems to work properly.


An autocrine Vitamin D-driven Th1 shutdown program can be exploited for COVID-19
Reuben McGregor, (22 others), Majid Kazemian and Behdad Afzali. 2020-07-19  (Preprint being edited for publication.)
https://www.biorxiv.org/content/10.1101/2020.07.18.210161v1

I regard this article as the most important article in the entire COVID-19 literature.

This is my best attempt to describe some complex processes I have no expertise in.

Th1 lymphocytes isolated from the lungs of patients with severe COVID-19 symptoms have an autocrine (https://vitamindstopscovid.info/02-autocrine/) signaling pathway which should be activated by high levels of complement (WP), to turn these cells off their initial hyper-inflammatory program which produces primarily pro-inflammatory IFNγ (interferon_gamma WP which has antiviral and anti-bacterial activity as well as stimulating inflammation: cell destruction such as by natural killer cells WP) and instead cause them to produce primarily the anti-inflammatory cytokine IL-10.  (The cells always produce both these cytokines, but this transition to a shutdown, anti-inflammatory program, involves less IFNγ and a lot more IL-10.)

However, this anti-inflammatory pathway is not working in the Th1 cells from patients with severe COVID-19, due solely to insufficient 25hydroxyvitaminD3 = 25OHD = calcifediol for each cell's autocrine signaling system to function.  (Until 2021-03-01 I mistakenly stated that the Th1 cells initially produced IL-17 - and that the experimenters restored the Th1's anti-inflammatory pathway by adding 25OHD in-vitro.)

This is a molecular and cellular explanation for why people with low vitamin D have wildly dysregulated, overly-inflammatory (cell killing), self-destructive immune responses.  Such responses drive sepsis, severe influenza, Kawasaki disease (KD WP), Multisystem Inflammatory Syndrome (MIS discussion) and of course severe COVID-19.  (See Paul Marik's explanation https://www.evms.edu/covid-19/covid_care_for_clinicians/ of how it is the immune response, not the virus, which causes the escalation to severe symptoms and death.  See https://aminotheory.com/cv19/#2015-Stagi for research which shows KD children have very low 25OHD vitamin D levels.)

In severe COVID-19, severe inflammation in the lungs damages endothelial cells (the inner lining of blood vessels and capillaries WP) leading to hypercoagulative blood, causing microembolisms and larger clots all over the body, which cause most of hypoxia, lasting harm and death.

It is not known whether the cause of all the hyper-inflammatory immune system dysregulation - which causes some COVID-19 sufferers people to develop severe symptoms - is primarily the failure of these Th1 lymphocytes to switch from being pro-inflammatory to anti-inflammatory, or whether this endothelial cell destruction etc. is also driven to a significant degree by similar failures in the autocrine signaling systems of many other types of regulatory and/or directly anti-pathogen immune cell.  However, the determination of the exact mechanism of failure in Th1 cells, in the context of such failures likely also occurring in other cell types, is an extraordinarily valuable contribution which needs to be very widely understood.

Low vitamin D levels (low circulating 25OHD, produced in the liver from UV-B-generated and/or ingested vitamin D3 cholecalciferol) are well known to reduce the effectiveness of numerous direct, anti-pathogen, responses by the innate immune system cells and to hinder the creation of antibodies for adaptive immune responses.  These immune functions of vitamin D 25OHD are due to it being needed, in the circulation, at higher levels than are sufficient for bone health (sufficient for the kidneys to produce their much lower concentration of circulating - and so hormonal - 25OHD), to supply the autocrine / paracrine (inside the cell / to nearby cells) signaling systems of all types of immune cells.  All types of immune cell can express the vitamin D receptor - and this is for autocrine/paracrine signaling - not for responding to the much lower levels of circulating 1,25OHD which regulates calcium-bone metabolism. https://vitamindstopscovid.info/02-autocrine/#02-nothorm .

See http://aminotheory.com/cv19/#2020-Fabbri [B] for why 40ng/ml or more 25OHD is required for these autocrine signaling systems to function properly.  See also the Quraishi et al. graph https://vitamindstopscovid.info/02-autocrine/#04-quraishi which suggests that innate immune cell responses which fight bacterial and perhaps fungal infections keep improving, presumably due to faster and stronger autocrine/paracrine signaling, as 25OHD levels rise, up to about 50ng/ml.

Please also see https://vitamindstopscovid.info/04-calcifediol/ for using a small, single oral dose of oral calcifediol (25OHD) plus D3 as the best treatment for hospitalised COVID-19 patients, since this raises circulating 25OHD to the levels needed for autocrine / paracrine signaling in a few hours, rather than in the several days to a week with vitamin D3.

For a more detailed summary of the McGregor et al. article, please see https://aminotheory.com/cv19/icu/#2020-McGregor .

Very strong clinical evidence of the importance of rapidly raising circulating 25OHD levels hospitalised COVID-19 patients can be found the Cordoba calcifediol (25OHD) RCT: Castillo et al. 2020: https://aminotheory.com/cv19/#2020-Castillo .


This McGregor et al. article is a beauty.  It gets down to brass tacks with the molecular processes of one aspect of the cytokine storm of immune dysregulation which is crucial to the development of severe COVID-19.  This failure may be the the biggest single pathological process which causes some people to develop severe COVID-19 symptoms.  If not, then the similar failures of autocrine signaling systems in all other immune cells would be the primary explanation.

This is one example of a specific vitamin D autocrine signaling system in one particular type of immune cell.  The following article discusses the evolutionary basis and other details of 189 human genes know to be regulated in an autocrine / paracrine manner by vitamin D in monocytes [WP], which are a subset of leukocytes [WP], which are a subset of immune cells which are a subset of the cell types in the body which use their own particular version of vitamin D autocrine / paracrine signaling. 

Primary Vitamin D Target Genes of Human Monocytes 
Veijo Nurminen, Sabine Seuter and Carsten Carlberg
Frontiers of Physiology 2019-03-15
https://www.frontiersin.org/articles/10.3389/fphys.2019.00194/full

I don't want to imply that I have read this, or that I would be able to understand it without weeks of work.  I cite it as an easy way of expanding upon this one concrete example which surely plays a crucial role in severe COVID-19 (and see https://aminotheory.com/cv19/#2020-Castillo for how oral 25OHD = calcifediol for hospitalised patients causes most of them to get much better, very quickly) to indicate how important this general principle of vitamin D autocrine signaling is.  

Why so complex?

Autocrine signaling is quite complex.  Inquiring minds want to know why this evolved and is used for so many cell types.  This is a Mouse Trap (video) approach to biology - Rube Goldberg [WP] engineering when we can imagine something simpler would do the job.  This wacky complexity is a feature of biology - and some or many of the idiosyncratic features of the evolved systems give rise to valuable mechanisms.  



This photo is of the San Francisco based Life Size Mousetrap of Mike Perez, which unfortunately seems not to have been active since 2013: http://lifesizemousetrap.org  This makes my 33 metre Sliiiiiiiiinky seem like child's play: https://www.firstpr.com.au/slinky/ .

Why, for instance, doesn't the sensing of the changed condition lead to direct upregulation and downregulation of whatever genes the cell needs to produce (or no longer produce) the proteins which will make respond as it is meant to? 

Sidebar for the really curious:

I guess the answer might be that the evolved capacity of the activated vitamin D receptor (a single VDR molecule bound to a 1,25OHD molecule) to upregulate and downregulate multiple genes turned out be flexible and useful.  The activated receptor complex binds to particular gene transcription promoter patterns (VDREs) in the DNA which are upstream of the genes whose rate of copying to messenger RNA is to be up- or down-regulated. 

This flexibility and ability to alter multiple genes at once may have some advantages over the simpler arrangement of, for instance, whatever signaling system enables an activated toll-like receptor [WP] to alter gene transcription (a first step in autocrine signaling) somehow evolving flexibility over multiple types of cell to alter as many genes in any one cell type, and in so many cell types, as are altered by the activated vitamin D receptor.

Just to keep us on our toes, in his article, Martin Hewison describes a separate set of processes operating in parallel to this autocrine signaling system, also driven by the activation of the toll-like receptors, which turns on some other aspects of the cell's response.

See also Martin Hewison's article
PMC2854233 regarding how the vitamin D autocrine signaling we humans have is specific to primates, and not found in rodents and other families of mammals.  He estimates this approach to autocrine signaling is about 40 million years old.

Please also see this article suggesting some hypotheses about the long-term evolutionary history of the vitamin D compounds and their receptors, enzymes and  binding proteins.

Evolutionary Origin of the Interferon–Immune Metabolic Axis: The Sterol–Vitamin D Link
Harry Newmark, Widad Dantoft and Peter Ghazal, Frontiers in Immunology, Molecular Innate Immunology 2017-02-09
https://www.frontiersin.org/articles/10.3389/fimmu.2017.00062

I roughly understood it up to about page 6.



© 2020 and 2021 Robin Whittle   Daylesford, Victoria, Australia