Vitamin D autocrine signaling - illustrated explanation

Robin Whittle rw@firstpr.com.au  31 December 2020 
(First established 2020-11-23.)

../ To the main page of this site.

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 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.   If you spot any errors or can suggest any improvements, please let me know.


Contents

#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.


#01-compounds

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

One definition of the term "vitamin D" is to refer only to D3 cholecalciferol and/or D2 ergocalciferol.  These are essential nutrients except that D3 can be made by UVB skin exposure.   D2 is inferior to D3 so I won't mention it further.   In this terminology, the two compounds created in the body from D3 are not referred to as "vitamin D".   However, they have "vitamin D" as part of their chemical names: 25-hydroxyvitaminD and 1,25dihydroxyvitraminD (sometimes with a 3 on the end).

I follow common usage and use the term "vitamin D" to refer collectively to the three compounds of interest: D3, 25OHD and 1,25OHD.



D3 cholecalciferol. [W]   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, 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 UVB light to break a ring between carbon 9 and 10. 



25OHD calcifediol = 25 hydroxyvitaminD3 = cacldiol [W].  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.

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 [W] = 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, paracrine and endocrine (hormonal) signaling

Firstly, a description of the one hormonal function of the vitamin D compounds.  A hormone is a compound in circulation, whose level (concentration)

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 (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 10 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 - failure of the bones to grow strong and straight.

So at healthy levels such as 40 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 hormonal 1,25OHD is the best known role of the vitamin D compounds, it 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 0.012ng/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:
There is room for improvement in the vitamin D research literature, including resolution of the following problems:
Circulating 1,25OHD is the one hormonal function of the vitamin D compounds.  A hormone [WP] is a compound which performs signaling between cells, over the whole body, by being transported everywhere in the bloodstream.  This is endocrine signaling AKA hormonal signaling.

Autocrine signaling [WP] occurs entirely within a particular cell, and so is unrelated to hormones or endocrinology.  It is not used to regulate steady-state or slowly changing processes - as is usually the case with hormonal signaling - but to enable an individual  cell to respond rapidly and fully to particular conditions.  Vitamin D autocrine signaling involves some external conditions causing 25OHD to be converted into 1,2OHD within the cell, which activates vitamin D receptors within the cell (or perhaps the cell's own receptors, but embedded in the cell membrane and binding with 1,25OHD outside the cell, where that 1,25OHD has been created from 25OHD inside this cell), some of which migrate to (or at least passively diffuse into) the nucleus [WP] and alter gene expression [WP], thereby causing this particular cell to respond to the new circumstances.  25OHD is consumed in this process.  This 1,25OHD is functioning as an autocrine agent.

Paracrine signaling [WP] is an extension of autocrine signaling in which some of the 1,25OHD diffuses out of the cell and into the interstitial fluid between cells, where it can affect the behaviour of other cells (probably different types of cell from the one in which the 1.25OHD is created) which are nearby.  This does not occur by this 1,25OD being transported long distances via the bloodstream.  Here, the 1,25OHD is functioning as a paracrine agent.  (Juxtracrine signaling is like paracrine signaling, except that the diffusion is only to adjacent cells.)

In one case at least (I can't remember which type of immune cell) the cell produces 1,25OHD as described for autocrine signaling but seems to have little or no vitamin D receptors in itself.  So it seems to be producing it purely for paracrine signaling to adjacent cells of different types,

Sidebar on intracrine vs. autocrine:

In the past the term "intracrine" was used alongside "autocrine" with the former meaning what we now know as "autocrine" in most respects, and "autocrine" meaning the same thing but with the receptor being on the outside of the same cell, rather than inside the cell.  As far as I know, hardly anyone cares about this distinction in 2020 and "autocrine" covers both receptor locations.  A good introduction to autocrine signaling from 2010, in which the term "intracrine" is used instead, is Martin Hewison's Vitamin D and the intracrinology of innate immunity  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854233/

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.


#03-minlev

At least 40ng/ml 25OHD blood levels required for good immune system function

The importance of proper (at least 40ng/ml = 100nmol/L = 1 part in 25 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/ and by this recent review article:

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.  I am not sure to what extent anyone has surveyed such genes in cell types normally found in tissues, and which are not normally circulating in the blood.

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

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 conversion 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 it 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 and average levels 1/5 or even as low as 1/10th of this.  50ng/ml is only one part by mass of 25OHD per 40,000,000 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, most of which is converted to 25OHD in the liver, to maintain this healthy level.


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:


  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:



  2. Is pointing in exactly the right direction for it to fit.  This needs to be correct in 3  dimensions too.

  3. Is rotated correctly - this is 1 dimension.
When this happens, the positive and negative changes 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 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.

    (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 slight effect - and I consider it insignificant.  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 and bolus D3 ../01-supp/#25plusD3 as well as other techniques.

You now have an understanding of some 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.


#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 highly regarded, and those who do it are morbidly obese, but it seems crazy to me when they should first try suitably high D3 intakes and all other measures to improve their health and reduce their life-threatening 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 - to reduce their obesity.

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:



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 responses are functioning properly.  The failures are due to weak immune responses which directly combat pathogens, 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.  This failure either 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 0.03, this is due to autocrine signaling not working properly in some - probably many - types of immune cell.  The 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 levels in the graphs pretty much directly 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.

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.


#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 signaling which assumes an interest in cell biology, but little prior knowledge.  In this example from 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) 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, 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, 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) 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 40ng/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).  

There, some subset of the activated receptor complexes find their way (by diffusion, I guess) to another molecule (retinoid X receptor [WP], not shown in these diagrams) with binds to them as well and the entire heterodimer 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.

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, 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 stops producing a pro-inflammatory cytokine and starts producing an 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, 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.


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 account of it, at:

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

and below.

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 et al. 2020-07-19
https://www.biorxiv.org/content/10.1101/2020.07.18.210161v1

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

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

This example concerns CD4 [W] (T-helper cells, T4 cells) which emit various cytokines to signal to other types of immune cells actions which destroy pathogens directly, destroy infected cells (cytotoxic - and so inflammatory and potentially self-destructive if not properly controlled) or take other actions.

Our Th1 (T Helper 1) cells start off on a pro-inflammatory program, referred to in the article as T-effector.  In this mode, they produce the inflammatory cytokine IFN-γ interferon gamma [W].

The autocrine signaling system of Th1 lymphocytes has evolved so that when it is activated and works properly, the Th1 cell switches to a second mode of operation (program) in which it instead emits the anti-inflammatory cytokine IL-10 [W].  The above article discusses how this does not happen in Th1 cells from hospitalised COVID-19 patients, due to lack of 25OHD while it works fine in TH1 cells from healthy controls.  (Unfortunately the authors do not report blood 25OHD levels from these patients.  The corresponding author wrote to me that they did not have access to any such measurements.)

"Complement" here refers to a number of proteins which form an important part of immune system signaling [W].   High levels of complement occur in the lungs with severe COVID-19.

The switch from effector T cells, important for pathogen clearance, into IL-10 producing cells reduces collateral damage and is a natural  transition in a T cell’s life-cycle. This suggests that IL-10 is produced by cells that are successfully transitioning into the Th1 shut down program. . . . We asked whether prolonged Th1 responses in COVID-19 patients are due to the failure in initiating this Th1 shutdown program.

This 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.

The high levels of complement activate the CD46 [WP] transmembrane receptor.  This induces (leads to more of) 24 transcription factors [WP].  Transcription factors are molecules which bind to specific locations on the DNA of chromosomes where they enhance or reduce (turn on or off) the transcription of particular genes into messenger RNA [WP].  The exact mechanisms of these transcription factors depend on epigenetic [WP] changes to the DNA, which would be specific to the particular cell type.  Two of these transcription factors upregulate the mRNAs for the vitamin D receptor [WP] (hereafter VDR) and for the CYP27B1 enzyme, (which I referred to above as the 1-hydroxylase enzyme, though its real name is more elaborate than this) which adds a hydroxyl group to the 1 position of 25OHD, converting it to 1,25OHD. 

In the vitamin D autocrine signaling system of other cell types, some other types of receptor would be activated initially, in response to some other stimulus than high levels of complement.  This would result in the induction of different transcription factors - and in some cases, some of these first set of messenger molecules in the cell do part of the work required for the cell to respond properly to the stimulus.  However, two of those transcription factors would again be for the genes for VDR and the CYP27B1 enzyme, just as in this particular example and the one above.

The next few steps are for this example, and more generally for the vitamin D autocrine functions of other cell types.

These two sets of mRNAs migrate from the nucleus and (for simplicity ignoring any post-translational modifications) find ribosomes in the cytosol [WP], which work their way along each mRNA and produce a protein according to the instructions encoded in the mRNA.  So, (ignoring any other processing steps) the cytosol now contains a lot more VDR and CYP27B1 enzyme molecules than it did before.

The next step is what should happen, but did not happen or did not happen enough in the Th1 cells isolated from the lungs of hospitalised COVID-19 patients.  

25OHD from the bloodstream migrates into the the interstitial fluid [WP] and from there it migrates into the cytosol of the Th1 lymphocyte.  The initial and continuing 25OHD levels in the cytosol of our TH1 cells are determined primarily or solely by passive diffusion from the concentration in the blood. 

By random bouncing around due to thermal motion, a 25OHD molecule finds its way into the active site of a CYP27B1 enzyme molecule, which, with co-factors, attaches an OH hydroxyl group onto it and releases it as 1,25OHD calcitriol.

The newly created 1,25OHD bounces around in the cytosol, as do all small molecules, until it finds itself correctly oriented in the binding site of a VDR molecule, to which it has a very strong affinity.  This pairing is the "activated" state of the VDR.  The VDR molecule changes its behaviour and the entire complex of VDR and its bound ligand, 1,25OHD, migrates to the nucleus, where it binds to a retinoid X receptor molecule and the whole heterodimer complex finds its way to particular parts of the DNA upstream of genes which are to be copied into messenger RNA molecules, as described above (VDRE).  The particulars of which genes these are differ between the various cell types which use vitamin D autocrine signaling.

The resulting new mRNAs (and potentially reductions in previously produced mRNAs) are worked on by ribosomes and so result new proteins (or less of of other proteins) in the cytosol which cause the cell to complete the action for which this autocrine signaling system has evolved.

In this particular example, the cell's actions includes shutting down production of pro-inflammatory IFN-γ production and ramping up production of inflammatory IL-10.

This does not occur to the degree it should in the cells the researchers isolated from the lungs of COVID-19 patients - but it did when they added sufficient 25OHD for the CYP27B1 enzyme to work on.

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 / paracrene manner by vitamin D in monocytes [WP], which are a subset of leucocytes [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 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 singe 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 Robin Whittle   Daylesford, Victoria, Australia