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3 Abnormalities in Genomic and Non-genomic Calcitriol/VDR Actions in CKD

3 Abnormalities in Genomic and Non-genomic Calcitriol/VDR Actions in CKD

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3 Molecular Biology of Vitamin D: Genomic and Nongenomic Actions of Vitamin D



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Fig. 3.2 Transcriptional regulation by the calcitriol/VDR complex. Multiple protein-protein and

protein-DNA interactions upon ligand binding to the VDR that facilitate the recruitment to the

transcriptional initiation complex of basal transcription factors (B) and coregulators (Co-reg) that

mediate vitamin D regulation of mRNA and/or micro RNA (mi-RNA) expression



(summarized in Fig. 3.2) upon ligand binding, the VDR molecule undergoes a conformational change that facilitates heterodimerization with the retinoid X receptor

(RXR) and the binding of the VDR/RXR complex to vitamin D responsive sequences

(VDREs) on the promoter regions of vitamin D responsive genes [27] to induce/

repress their expression. There is variability in VDRE sequences, but those associated with the highest affinity for VDR consist in two direct imperfect repeats with a

spacer of 3 nucleotides (DR3). Chromosome conformation capture technology has

demonstrated multiple simultaneous rather than a single site for binding of the

ligand-activated VDR/RXR complex within 100 Kb either 5′ or 3′ from the transcription start site of a target gene [28]. Chromatin looping juxtaposes distal and

more proximal VDREs thus facilitating the simultaneous recruitment of basic transcription factors, co-activator and/or co-repressor molecules to multiple VDR-RXR/

VDRE complexes, as demonstrated for calcitriol potent induction of the RANKL

gene, critical for calcitriol-driven osteoclastogenesis and bone resorption [29].

Similar single or super-complexes of VDR/RXR bound to DNA transcriptionally

activate/repress the expression of the 500 to 1,000 genes that regulate a healthy

aging, and consequently, the survival benefits of a normal vitamin D status. The

most characterized of these calcitriol/VDR genomic actions include the suppression

of PTH synthesis, induction of the phosphaturic hormone FGF23, the longevity

gene klotho, the calcium channel TRPV6 in enterocytes, the rate limiting step in

intestinal calcium absorption, the parathyroid calcium sensing receptor or of the



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receptor of the canonical Wnt pathway LRP5 in bone, all essential effectors for

normal skeletal development and mineralization, as thoroughly reviewed in [27].

In addition, VDRE sequences for VDR/RXR binding have been found in gene

promoters that drive the expression of micro RNAs (Fig. 3.2) [30]. Micro RNAs are

short (18–25 nucleotides) non-coding RNAs that control the expression of 30 % of

the genes in the genome through binding to the 3′ untranslated region (UTRs) of

target mRNA decreasing either mRNA stability or protein translation. An example

is calcitriol upregulation of microRNA-145 (miR-145), a recently identified mechanism for calcitriol suppression of proliferation in gastric cancer [31]. Importantly,

miR-145 is the most abundant microRNA in normal vascular smooth muscle cells

and it is downregulated in proliferative vascular diseases [32] suggesting a role for

calcitriol induction of miR-145 in the vascular protective actions of calcitriol and

analogs.

Additional mechanisms for calcitriol/VDR regulation of microRNA levels

include the control of the microRNA processing machinery or of the chromatin

opening upon VDR binding to increase the accessibility for transcription factors

that induce/repress micro RNA transcription [33]. In fact, calcitriol upregulates

KHSRP and TARDBP, two proteins involved in the biogenesis and maturation of

micro RNA in colon cancer cells [34].

Several “non-classical” calcitriol/VDR genomic actions involve an indirect control of the expression of an apparent target through the transcriptional control of an

essential inducer or repressor. Examples include: (a) Calcitriol induction of the

growth suppressor p27 through direct transrepression of p45(Skp2) which bindsp27to induce its proteosomal degradation [35]; (b) Calcitriol suppression of

ADAM17 gene expression to control parathyroid hyperplasia, through the induction

of C/EBPβ [36]; (c) Calcitriol 30-fold induction of FGF23, which is markedly attenuated by cycloheximide, an inhibitor of new protein synthesis. This suggests that, in

addition to a mild direct induction of the FGF23 gene, calcitriol suppression/induction of a yet unknown mediator is required for the full transactivation of the FGF23

gene [37].

In addition, the calcitriol/VDR complex binds transcriptional regulators and

modifies their transcriptional activity, in signaling pathways unrelated to vitamin D

biological actions, as demonstrated for VDR inhibition of Wnt activation through

physical VDR interactions with β-catenin outside bone [38]. Also, direct proteinprotein interactions of calcitriol/VDR complexes with Sirt1 and FOXO proteins,

which control aging processes, which modify their cellular function prolonging survival [39].

The calcitriol/VDR complex also affects the rates of mRNA translation. This

is a highly needed process when cells need to mount acute responses upon life

threatening conditions such as starvation or strong growth signals. These vitamin D actions include both suppression of mTOR as demonstrated in breast cancer [40] and induction of autophagy, through direct upregulation of essential

autophagy genes, as demonstrated for VDR protection from immune dysfunction of the intestinal barrier by Paneth cells, thus maintaining the homeostasis of

the microbiota [41].



3 Molecular Biology of Vitamin D: Genomic and Nongenomic Actions of Vitamin D



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Calcitriol also exerts rapid “non-genomic” actions occurring within minutes of

exposure to calcitriol. Some of these less characterized rapid actions also involve

the cytosolic VDR, although other potential receptors have been identified [12].

These rapid actions regulate intracellular calcium fluxes, the degree of protein

phosphorylation, acetylation and subcellular localization which, by affecting protein function, greatly modify classical and non-classical direct and indirect

genomic signals (Reviewed in [12]). An emerging relevant field is the impact of

rapid vitamin D actions on pathways regulating the stability and/or processing of

microRNAs [34].

For most biological actions, the intracellular levels of both calcitriol and VDR

determine the magnitude of calcitriol/VDR complex formation and with it, the efficacy for direct or indirect gene transactivation/transrepression by the 1,25D/VDR

complex, and both are reduced in CKD [17]. The development of calcitriol analogs

that selectively maintain the benefits of VDR activation with less calcemic or phosphatemic activity [42] has helped therapeutically. They allow compensating the

resistance to therapy caused by CKD-induced VDR reductions through safer escalation of analog dosage. However, therapies with high doses of calcitriol or its analogs

could reduce the levels of intracellular calcitriol (analog) available to bind VDR by

inducing CYP24A1 (See Fig. 3.1). This enzyme, responsible for calcitriol (analog)

degradation, is constitutively expressed in the kidney, and strongly induced by

either calcitriol or its analogs in every vitamin D target tissue to avoid/reduce the

toxicity associated to an excess of active vitamin D [27].

Decreases in cellular levels of the VDR partner for its genomic actions, the retinoid X receptor (RXR), as well as uremic toxin-induced reductions of 1,25D/VDRRXR binding to DNA, further impair the response of these patients to vitamin D

therapy (Reviewed in [43]).

Figure 3.1 also presents a previously unrecognized synergy between 25(OH)D

and calcitriol for VDR activation that could be exploited to safely improve clinical

outcomes in CKD without increasing calcitriol doses. Studies in the CYP27B1 null

mouse [44], which lacks the enzyme that converts 25(OH)D to calcitriol, and also

in vitro, using 25(OH)D analogs chemically modified to prevent hydroxylation at

carbon 1[45, 46], have demonstrated that 25(OH)D can not only activate the VDR

directly, but more importantly, it can synergize with calcitriol activation of the

VDR. As will be discussed below, this synergy is sufficient to overcome the parathyroid resistance to low doses of calcitriol (or its analogs) caused by VDR reductions and accumulation of uremic toxins even in advanced CKD [36]. Importantly,

this safe synergy can be achieved simply by ensuring normal serum 25(OH)D

through nutritional vitamin D supplementation [36].

The ubiquitously distributed 25-hydroxylases are not as tightly regulated as the

constitutive renal CYP27B1 and CYP24A1, thus providing a safe alternative to

enhance survival by counteracting with local 25(OH)D synthesis the reductions in

intracellular levels of the calcitriol/VDR complex induced by CKD. A better understanding of the modulators of the tissue specific expression and activity of

25-hydroxylases in CKD could improve current recommendations to enhance the

survival benefits of a normal vitamin D status in these patients.



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A.S. Dusso



The next section will update our understanding of the most critical vitamin D

actions to attenuate CKD progression and improve clinical outcomes:

1. Suppression of PTH synthesis, parathyroid hyperplasia and the onset of vitamin

D resistance;

2. Maintenance of skeletal and vascular integrity unrelated to the attenuation of

secondary hyperparathyroidism;

3. Induction of bone FGF23 production and renal klotho content to prevent/attenuate hyperphosphatemia;

4. Downregulation of systemic disorders that reduce renal klotho;

5. Induction of anti-aging actions unrelated to maintenance of renal klotho;

For each of these actions critical for survival, this review will examine:

(a) What is indisputably known on the subject at the molecular level?

(b) What are the findings from the last 5 years that have challenged our current

understanding of the pathogenesis of the pro-aging disorder?

(c) How might the new knowledge impact clinical practice regarding both, the

safety of current therapeutic strategies with vitamin D and the accuracy of commonly used biomarkers?



3.4



Vitamin D Suppression of PTH Synthesis, Parathyroid

Hyperplasia and the Onset of Vitamin D Resistance



Near all patients with end stage renal disease develop SHPT. Because of the severe

adverse impact of SHPT on morbidity and mortality, the parathyroid gland is one of the

best studied targets of vitamin D actions. Figure 3.3 summarizes the multiple calcitriol/

VDR actions involved in the suppression of PTH synthesis and hyperplastic growth.

Hypocalcemia, hyperphosphatemia and vitamin D deficiency are the main causes

of SHPT [47]. The calcitriol/VDR complex suppresses PTH synthesis through a

direct binding to a “classical” negative VDRE on the PTH gene promoter [27].



Fig. 3.3 Vitamin D regulation of parathyroid hyper function in CKD



3 Molecular Biology of Vitamin D: Genomic and Nongenomic Actions of Vitamin D



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Vitamin D and/or calcitriol deficiency also impair the response of the parathyroid

gland to calcium due to reductions in parathyroid content of the calcium sensing

receptor (CaSR), as demonstrated in vitamin D deficient rats [48] and CKD patients

[49]. In fact, the CaSR gene is directly induced by calcitriol through VDR/RXR

binding to VDREs in this gene promoter [50].

Calcitriol induction of FGF23 synthesis in bone cells of the osteoblastic/osteocyte lineage provides an additional indirect mechanism for vitamin D suppression

of PTH secretion and parathyroid hyperplasia, provided there is sufficient parathyroid klotho [51], another gene induced by the calcitriol/VDR complex [27].

The prolonged persistence of hypocalcemia or vitamin D deficiency induces parathyroid cell proliferation to meet the requirements for higher serum PTH to normalize serum

calcium. Also, hyperphosphatemia directly stimulates parathyroid hyperplasia [52]. The

severity of parathyroid cell growth determines not only higher serum PTH but also

marked reductions in parathyroid VDR, CaSR, FGF receptors, and cell membrane klotho,

which impair PTH suppression by active vitamin D, oral calcium or increases in FGF23.

The reports of increased TGFα in parathyroid adenomas and in diffuse and nodular glands from CKD patients [53] were critical to identify the essential role of

TGFα activation of its receptor, the EGF receptor (EGFR), in the severity of parathyroid hyperplasia and VDR reductions [54]. Briefly, as summarized in Fig. 3.4,

the release of mature TGFα from its transmembrane precursor by ADAM17, an

enzyme essential for parathyroid gland development [36], initiates a powerful



Fig. 3.4 Pathogenesis of parathyroid hyperplasia and VDR reduction in CKD. The vicious

ADAM17/TGFα-EGFR cycle for exacerbated parathyroid growth and VDR reduction is safely

and effectively counteracted by synergistic interactions between 25(OH)D and calcitriol through

the induction of C/EBPβ



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