Serum concentrations of 1,25-dihydroxyvitamin D3 in Platyrrhini and Catarrhini: A phylogenetic appraisal

We measured the serum concentration of 25-hydroxyvitamin D₃ (25-OH-D₃) and 1,25-dihydroxyvitamin D₃ (1,25-[OH]₂-D₃) in 23 different Platyrrhines from four different genera and in 21 Catarrhines from six different genera in residence at the Los Angeles Zoo. The mean (±S.E.) serum concentration of 1,25-(OH)₂-D₃ was significantly greater in Platyrrhines (810 ± 119 pg/ml) than in Catarrhines (61 ± 5 pg/ml), suggesting that high circulating concentrations of the active vitamin D hormone were a characteristic of New World primates in both the Cebidae and Callitrichidae family. This increase in the serum concentration of 1,25-(OH)₂-D₃ is probably an adaptational response on the part of Platyrrhini to offset a relative decrease in the concentration of specific receptor for 1,25-(OH)₂-D₃ in target tissues for the hormone.     

Metabolism of 1alpha-hydroxyvitamin D3 to 1alpha, 25-dihydroxyvitamin D3 in perfused rat liver.

The metabolism of 1α-hydroxyvitamin D₃ (1α-OH-D₃) was studied in rat liver perfused with [³H]-1α-OH-D₃. [³H]-1α-OH-D₃ was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D₃ [1α,25-(OH)₂-D₃] by co-chromatography with synthetic 1α,25-(OH)₂-D₃ as well as by gas chromatography-mass spectrometry. [³H]-1α,25-(OH)₂-D₃ appeared in the perfusate as early as 20 min after addition of [³H]-1α-OH-D₃, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D₃ is metabolized to 1α,25-(OH)₂-D₃, which exerts biological effects on bone and intestine.     

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)₂D₃ in primary cultured murine renal tubules.Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD₃ (10⁻⁶ M), 1,25-(OH)₂D₃ was determined. PTH(1-34) stimulated the conversion of 25-OHD₃ to 1,25-(OH)₂D₃, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1α-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1α or 24-hydroxylase mRNA.These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)₂D in patients with severe primary hyperparathyroidism with renal failure.     

Effect of 1,25-dihydroxyvitamin D3 on human keratinocytes grown under different culture conditions

Summary 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂-D₃) is known to decrease the proliferation and increase the differentiation of different cell types including human keratinocytes. The growth and differentiation of keratinocytes in the presence of 1,25-(OH)₂-D₃ using serum-free media formulations has been described previously. This investigation extends these studies to describe various culture conditions with human foreskin keratinocytes to determine the optimal antiproliferative activity of 1,25-(OH)₂-D₃. Keratinocytes were plated onto tissue culture dishes using one of three basic serum-free media protocols; a) with no feeder layer in keratinocyte growth medium (KGM); b) onto mitomycin C-treated 3T3 mouse embryo fibroblasts; or c) onto mitomycin C-treated dermal human fibroblasts. The last two protocols utilized Dulbecco's modified Eagle's Medium (DMEM) supplemented with growth factors. Keratinocyte cell growth was greatest in the KGM medium. Although the growth of keratinocytes on either feeder layer was similar, there were differences in the ability of the cells to form envelopes in the presence of 1,25-(OH)₂-D₃. The addition of hydrocortisone and cholera toxin to the medium also affected the response of the keratinocytes to 1,25-(OH)₂-D₃. The antiproliferative effect of 1,25-(OH)₂-D₃ was not altered by varying the extracellular calcium levels from 0.25 to 3 mM. The antiproliferative activity of 1,25-(OH)₂-D₃ is attenuated in cells at low density. Our results suggest that an optimal condition to investigate the ability of 1,25-(OH)₂-D₃ to inhibit keratinocyte proliferation is at preconfluent cell density in the presence of KGM supplemented with 1.5 mM calcium without a feeder layer. These conditions are not appropriate for investigating the enhancement of differentiation by 1,25-(OH)₂-D₃, but can be used to assay other agents that modulate keratinocyte proliferation. Portions of this work were presented and abstracted at the April 1988 meeting of the Society of Investigative Dermatology (J. Inv. Derm. 90(4): 586; 1988) and the February 1988 meeting of New York Academy of Sciences (Ann NY Acad. Sci. 548: 341–342; 1988).     

Failure of 1, 25 dihydroxycholecalciferol (1, 25-(OH) 2-D3) to modify cyclic AMP levels in parathyroid hormone-treated and untreated bone cells.

Treatment with 1, 25-(OH) 2-D3 (1 ng/ml-25 ng/ml) for periods ranging from 2.5 min. to 60 min. did not alter cyclic AMP levels in bone cells isolated from periosteum-free rat calvaria, or in cells isolated from rat periosteal tissues. 1, 25-(OH) 2-D3 failed to modify the acute increases in cyclic AMP elicited by PTH (10 ng/ml-1 ug/ml). Two separate 1, 25-(OH) 2-D3 preparations, biologically active in other systems, were ineffective under a wide variety of experimental conditions. These results suggest that 1, 25-(OH) 2-D3 is not an acute modulator of cyclic AMP metabolism in PTH-treated and untreated bone cells.     

Synthesis and biological evaluation of a 3-positon epimer of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], possesses a hydroxypropoxy substituent at the 2β-position of 1,25(OH)₂D₃. ED-71 has potent biological effects on bone and is currently under phase III clinical studies for bone fracture prevention. It is well-known that the synthesis and secretion of parathyroid hormone (PTH) is regulated by 1,25(OH)₂D₃. Interestingly, during clinical development of ED-71, serum intact PTH in osteoporotic patients did not change significantly upon treatment with ED-71. The reason remains unclear, however. Brown et al. reported that 3-epi-1,25(OH)₂D₃, an epimer of 1,25(OH)₂D₃ at the 3-position, shows equipotent and prolonged activity compared to 1,25(OH)₂D₃ at suppressing PTH secretion. Since ED-71 has a bulky hydroxypropoxy substituent at the 2-position, epimerization at the adjacent and sterically hindered 3-position might be prevented, which may account for its weak potency in PTH suppression observed in clinical studies. We have significant interest in ED-71 epimerization at the 3-position and the biological potency of 3-epi-ED-71 in suppressing PTH secretion. In the present studies, synthesis of 3-epi-ED-71 and investigations of in vitro suppression of PTH using bovine parathyroid cells are described. The inhibitory potency of vitamin D₃ analogs were found to be 1,25(OH)₂D₃ > ED-71 ≥ 3-epi-1,25(OH)₂D₃  3-epi-ED-71. ED-71 and 3-epi-ED-71 showed weak activity towards PTH suppression in our assays.     

Biphasic action of prostaglandin E2 on conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in chick renal tubules

The effect of PGE₂ on the conversion of 25-hydroxyvitamin D₃ (25 OH D₃) to 1,25-dihydroxyvitamin D₃ (1,25- (OH) ₂D₃) by isolated renal tubules from vitamin D deficient chicks was studied under a variety of experimental conditions. In the absence of added vitamin D metabolites, PGE₂ (2 × 10⁻⁶M) caused an immediate inhibition of formation of 1,25-(OH) ₂D₃, followed by a delayed stimulation, apparent after 15 h exposure to PGE₂. Pretreatment of the tubules with 1,25-(OH) ₂D₃ prevented the immediate inhibitory action of PGE₂, and allowed the stimulation to be apparent after 4 h exposure to PGE₂. The cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX) significantly stimulated the formation of 1,25-(OH) ₂D₃. PGE₂ significantly inhibited 1,25-(OH) ₂D₃ formation in tubules which had been stimulated by IBMX. PGE₂ stimulated the adenylate cyclase activity in a crude particulate fraction from the chick kidney, and raised cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) levels in the renal tubules.It is concluded that PGE₂ can either stimulate or inhibit 1,25-(OH) ₂D₃ formation in chick renal tubules. The stimulatory effect may be partly due to elevation of cyclic AMP. The mechanism of the inhibitory effect requires further investigation.     

Studies on calciferol metabolism: Metabolism of 25-hydroxy-Vitamin D3 by the chicken embryo

It is known that after birth of a vertebrate there is a requirement for the metabolism of Vitamin D₃ (cholecalciferol) to 1,25-(OH)₂-Vitamin D₃ to produce the hormonally active form essential for calcium homeostasis. However it is not known whether the enzymatic capability to produce 1,25-(OH)₂-D₃ only appears after birth or whether it is generated in the embryo. Presented in this paper are results of studies designed to measure the production and localization of 1,25-(OH)₂-D₃ in the embryo. It was found that the renal enzyme, 25-OH-cholecalciferol-1-hydroxylase, which is capable of producing 1,25-(OH)₂-D₃, is present as early as day 9 of incubation (12 days before hatch) in White Leghorn chicks. Further, the enzyme activity increases 6-fold to a maximal level which occurs on the day of hatching. 1,25-(OH)₂-D₃ was shown to be produced in vivo at day 17 and was found then in low levels in the embryonic intestine and kidney. Thus we have shown that 1,25-(OH)₂-D₃ is made by embryonic chick kidneys and is found in low levels in embryonic chick intestine and kidney significantly before hatch.     

Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3.

Kidney homogenates from chicks fed a vitamin D-deficient diet for 10 days and supplemented with 6.5 nmol of vitamin D₃ 48 hr prior to sacrifice metabolized $\text{in}\text{vitro}\text{[}{}^3\text{H}\text{]}$-25-hydroxyvitamin D₃ (25-OH-D₃) to 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified [³H]-24,25-(OH)₂-D₃, 3 similar metabolites (peaks A′, C′ and E′) were produced. On high pressure liquid chromatography, peaks A, C and E migrated to exactly the same respective positions as peaks A′, C′ and E′. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [³H]-25-OH-D₃ or [³H]-24,25-(OH)₂-D₃. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)₂-D₃ in the kidney of chicks supplemented with vitamin D₃.     

Simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D2 and D3 in human plasma application to nutritional studies

A simplified method for the determination of 25-hydroxy and 1α,25-dihydroxy metabolites of vitamins D₂ and D₃ in human plasma was developed. Plasma samples were deproteinizated and applied to a Bond Elut C₁₈ OH cartridge to separate 25-hydroxyvitamin D (25-OH-D) and 1α-25-dihydroxyvitamin D [1,25(OH)₂D] fractions. The 25-OH-D fraction was purified by a Bond Elut C₁₈ cartridge and 25-OH-D₂ and 25-OH-D₃ were assayed by HPLC using a Zorbax SIL column. The 1,25(OH)₂D fraction obtained above was subsequently applied to HPLC using a Zorbax SIL column to separate 1,25(OH)₂D₂ and 1,25(OH)₂D₃ fractions which were determined by a radioreceptor assay (RRA) using calf thymus receptor. The method was applied to nutritional studies.     

Immunoreactive parathyroid hormone levels in platyrrhini and catarrhini: A comparative analysis with three different assays

Serum concentrations of the hormonal form of vitamin D₃—1,25-dihydroxy-vitamin D₃ [1,25-(OH)₂-D₃]—are elevated in many genera of platyrrhines when compared to catarrhines; this elevation is presumed to result from a decrease in the ability of the target cell receptor effectively to recognize 1,25-(OH)₂-D₃. The activity of the renal 25-hydroxyvitumin D₃-1α-hydroxylase, the mammalian enzyme which synthesizes the majority of the circulating 1,25-(OH)₂-D₃, is accelerated by parathyroid hormone (PTH). In order to determine whether the elevated serum concentrations of 1,25-(OH)₂-D₃ in platyrrhines were the result of relative hyperparathyroidism, we measured serum levels of immunoreactive parathyroid hormone (iPTH) in normocalcemic platyrrhines, catarrhines, and human subjects with assays that recognize different domains of the human PTH molecule. Antisera directed against the biologically active, aminoterminus of PTH yielded comparable mean values for iPTH among three test groups. The mean concentration of iPTH as assessed by a “proximal” midregion assay was significantly reduced in platyrrhine serum when compared to either human or catarrhine serum. A “distal” midregion assay yielded a reduced mean value for iPTH in both platyrrhine and catarrhine serum when compared to human serum. These data suggest that 1) high circulating levels of 1,25-(OH)₂-D₃ in New World primates are not the result of hyperparathyroidism; and 2) structural homology between human and primate PTH diminishes progressively as one moves toward the carboxyterminus of the molecule and is lost more rapidly in the platyrrhine than in the catarrhine hormone.     

Autoradiographic studies with 3H 1,25 (OH)2 vitamin D3 and 3H 25 (OH) vitamin D3 in rat parathyroid glands

Summary After injection of radiolabeled 1,25 (OH)₂ vitamin D₃, nuclear concentration of radioactivity is observed in parenchymal cells of the parathyroid gland in pregnant, adult male, and 10-day male neonatal rats. In competition studies with unlabeled 1,25 (OH)₂ vitamin D₃, but not with 25 (OH) vitamin D₃, nuclear uptake is prevented. Experiments with ³H 25 (OH) vitamin D₃, in contrast to ³H 1,25 (OH)₂ vitamin D₃, do not show nuclear concentration in cells of the parathyroid. The results of the autoradiographic studies suggest the presence of receptors for a direct effect of 1,25 (OH)₂ vitamin D₃ on the parathyroid gland for modulation of parathyroid hormone secretion.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

A specific binding protein for 1,25-dihydroxyvitamin D3 in rat intestinal cytosol.

In the presence of 0.3 M potassium chloride and 0.5 mM dithiothreitol, rat intestinal cytosol contains two binding proteins for 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)¹ having sedimentation coefficients of 3.2S and 5–6S. The 3.2S protein is specific for 1,25-(OH)₂D₃ as determined by competition analysis, whereas the 5–6S protein binds 25-hydroxyvitamin D₃ (25-OH-D₃) exclusively.     

Circadian rhythm of 1 alpha,25-dihydroxyvitamin D3 production in egg-laying hens.

The activity of renal 25-hydroxyvitamin D₃(25-OH-D₃)-1α- and 24-hydroxylase and the plasma concentrations of vitamin D metabolites were investigated in relation to the ovulatory cycle in egg-laying hens. The time after ovulation was estimated from the position of the egg in the oviduct and the dry weight of the egg-shell. The $\text{in}\text{vitro}$ renal 25-OH-D₃-1α-hydroxylase activity was significantly enhanced 14–16 hr after ovulation, whereas 25-OH-D₃-24-hydroxylase activity remained unchanged. The plasma level of 1α,25-dihydroxyvitamin D [1α,25-(OH)₂-D] was also increased 14–16 hr after ovulation in accord with the enhancement of the renal 1α-hydroxylase activity. The plasma level of 24,25-dihydroxyvitamin D did not change during the ovulatory cycle. These results strongly suggest that 1α,25-(OH)₂-D₃ production in the kidney varies in a circadian rhythm during the ovulatory cycle in egg-laying hens.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

The endocrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)₂D₃ and PTH. This was investigated in ATDC5 cells treated with 10^?8 M 1,25(OH)₂D₃ or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)₂D₃ receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)₂D₃ decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)₂D₃ treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate. 1,25(OH)₂D₃ decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)₂D₃ by increasing VDR production. In light of the role of 1,25(OH)₂D₃ and PTHrP in modulating chondrocyte differentiation, 1,25(OH)₂D₃ in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.

     

Bone resorbing activities of 24-hydroxy stereoisomers of 24-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

R and S isomers of 24-OH-D₃ and 24,25-(OH)₂D₃ were tested for their effects on bone resorption $\text{in vitro}$. 24(R), 25-(OH)₂D₃ was more active than 24(S),25-(OH)₂D₃. Likewise, 24(R)-OH-D₃ was more active than 24(S)-OH-D₃. The bone resorbing activity of 24(R)-OH-D₃ was equivalent to that of 25-OH-D₃; 24(R),25-(OH)₂D₃ was somewhat less potent. The results indicate that there is discrimination between the isomers of these compounds at the level of the responding tissue.