1,25(OH)2D3 receptor regulation and 1,25(OH)2D3 effects in primary cultures of growth cartilage cells of the rat

Summary Vitamin D deficiency leads to disturbed calcification of growth cartilage and enlargement of growth plate, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of 1,25(OH)₂D₃ receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In primary cultures of tibial growth cartilage of male SD rats (80 g), specific binding of [³H]-1,25(OH)₂D₃ is noted in both the logarithmic growth phase and at confluence (N_max 12780 molecules/cell versus 4368 molecules/cell). Scatchard analysis revealed the presence of a single class of noninteracting binding sites. K_D was 10⁻¹¹ M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA cellulose affinity chromatography. In immunohistology, growth cartilage cells (rabbit tibia) expressed nuclear 1,25(OH)₂D₃ receptors most prominently in the proliferative and hypertrophic zone. This corresponds to binding data which showed highest N_max in the proliferating cartilage. 1,25(OH)₂D₃ in the presence of delipidated fetal calf serum (FCS) had a biphasic effect on cell proliferation and density, i.e., stimulation at 10⁻¹² M and dose-dependent inhibition at 10⁻¹⁰ M and below. Inhibition was specific and not seen with 24,25(OH)₂D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and effects of 1,25(OH)₂D₃ on chondrocyte proliferation point to a role of vitamin D in the homeostasis of growth cartilage.     

Effect of vitamin D on growth cartilage cell proliferation in vitro

Disturbed calcification of the growth plate and stunting is a frequent finding in vitamin D-deficiency rickets, vitamin D-dependency rickets and renal osteodystrophy, illustrating that chondrocytes are a target for vitamin D. This observation prompted an investigation of Ic, 25-dihydroxy vitamin D₃ [1,25(OH)₂D₃] receptor expression and action of vitamin D metabolites on chondrocyte proliferation. In tibial growth plates and in primary cultures of tibial growth cartilage of male Sprague-Dawley rats (80 g) specific binding of [³H]-1,25(OH)₂D₃ was noted. Scatchard analysis revealed the presence of a single class of non-interacting binding sites.K _d was 10^–11 M irrespective of growth phase. The binding macromolecule had a sedimentation coefficient of 3.5 S. Interaction with DNA was demonstrated by DNA-cellulose affinity chromatography. By immunohistology, growth cartilage cells (rabbit tibia) were shown to express nuclear 1,25(OH)₂D₃ receptors, most prominently in the proliferative and early hypertrophic zone. This corresponds to binding data which showed highest binding of 1,25(OH)₂D₃ in the logarithmic growth phase (12,780 molecules/cell versus 4,538 molecules/cell in confluent cells) in primary cultures of growth plate chondrocytes. In the presence of delipidated fetal calf serum 1,25(OH)₂D₃ had a biphasic effect on cell proliferation and density, i.e. stimulation at 10^–12 M and dose-dependent inhibition at 10^–10 M and below. Inhibition was specific and not seen with 24 (R), 25-dihydroxyvitamin D₃ or dexamethasone. Growth phase-dependent 1,25(OH)₂D₃ receptor expression and specific effects of 1,25(OH)₂D₃ on chrondrocyte proliferation in vitro point to a role for vitamin D in the homeostasis of growth cartilage of the rat.     

Thyroid hormone‐mediated growth and differentiation of growth plate chondrocytes involves IGF‐1 modulation of β‐catenin signaling

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β‐catenin signaling pathway. Insulin‐like growth factor 1 (IGF‐1) has been described as a stabilizer of β‐catenin, and thyroid hormone is a known stimulator of IGF‐1 receptor expression. The purpose of this study was to test the hypothesis that IGF‐1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β‐catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF‐1 and the IGF‐ receptor (IGF1R) stimulate Wnt‐4 expression and β‐catenin activation in growth plate chondrocytes. The positive effects of IGF‐1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T₃ activates IGF‐1/IGF1R signaling and IGF‐1‐dependent PI3K/Akt/GSK‐3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T₃‐mediated Wnt‐4 expression, β‐catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β‐catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF‐1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF‐1/IGF1R‐mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β‐catenin signaling, which is at least in part initiated by IGF‐1 signaling or the IGF‐1‐activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research     

Hormonal regulation of vitamin D receptor levels and osteocalcin synthesis in human osteosarcoma cells

1,25(OH)₂D₃ was found to regulate its own receptor levels via an increase in corresponding mRNA levels in human osteoblast-like osteosarcoma cells (MG-63). In addition, exposure of the cells for 24h to dexamethasone, estradiol, retinoic acid, or triiodothyronine resulted in a dose-dependent accumulation of hVDR mRNA. Combination of 1,25(OH)₂D₃ with any other hormone used in this study did not result in an additive increase in hVDR mRNA levels. Progesterone or dihydrotestosterone did not influence hVDR mRNA levels. Of the studied hormones, only 1,25(OH)₂D₃ was alone able to stimulate the synthesis and secretion of osteocalcin. Compared with 1,25(OH)₂D₃, the combination of 1,25(OH)₂D₃ and retinoic acid resulted an increased synthesis of osteocalcin. In contrast, the combination of 1,25(OH)₂D₃ with dexamethasone, estradiol, or triiodothyronine diminished the stimulatory effect of 1,25(OH)₂D₃. A complex interaction of several different hormone receptors seems to occur within the regulatory regions of hVDR and osteocalcin genes, or at the level of translation, resulting, in each case, a finely adjusted vitamin D receptor and osteocalcin expression.     

Suramin, hydrocortisone, and retinoic acid modify inhibitory effects of 1,25-dihydroxyvitamin D(3) on prostatic epithelial cells

The proliferation of prostatic epithelial cells is regulated by the complex interplay of numerous growth-stimulatory and growth-inhibitory factors. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has recently been identified as a potent inhibitor of the growth of prostatic epithelial cells. Epidemiologic studies indicate that vitamin D deficiency may be a risk factor for the development of clinical prostate cancer, possibly due to increased growth and reduced differentiation of prostatic cells in an environment with decreased 1,25(OH)(2)D(3). The application of vitamin D or analogs in chemotherapy against prostate and other cancers is being explored by several investigators. In order to use vitamin D most efficaciously in a clinical setting, it may be beneficial to learn more about the interaction of 1,25(OH)(2)D(3) with other factors that regulate prostatic epithelial cellular growth. In this study, we examined the effect of the proliferative status of cultured cells on their ability to respond to 1,25(OH)(2)D(3), and found that minimally proliferative cells were equally as responsive to 1,25(OH)2D3 as actively dividing cells. We noted no apparent interaction of 1,25(OH)(2)D(3) with epidermal growth factor, insulin-like growth factor, cholera toxin, or transforming growth factor-β, but we did find synergistic inhibitory effects of 1,25(OH)(2)D(3) with suramin and retinoic acid. Perhaps most noteworthy was the dramatic increase in potency of 1,25(OH)(2)D(3) that occurred upon deletion of hydrocortisone from the culture medium. Our in vitro studies indicate that combination therapy of vitamin D analogs with suramin, vitamin A analogs, or anti-glucocorticoids might be considered for prostate cancer.     

1,25(OH)2D3 and dihydrotestosterone interact to regulate proliferation and differentiation of epiphyseal chondrocytes

Growth plate chondrocytes are affected by 1,25(OH)₂D₃ and androgens, which may critically interact to regulate proliferation and differentiation during the male pubertal growth spurt. We investigated possible interactions of 1,25(OH)₂D₃ and the non-aromatizable androgen dihydrotestosterone (DHT) in primary chondrocyte cultures from young male rats. DHT and 1,25(OH)₂D₃ independently stimulated DNA synthesis and cell proliferation in a dose-dependent manner with maximally effective doses of [10^-8 M] and [10^-12 M], respectively. Both DHT and 1,25(OH)₂D₃ stimulated the expression and release of IGF-I, and the proliferative effects of each hormone were prevented by an IGF-I antibody. DHT and 1,25(OH)₂D₃ increased messenger RNAs (mRNAs) of their cognate receptors and of IGF-I receptor mRNA (IGF-I-R). 1,25(OH)₂D₃ also stimulated mRNA of the androgen receptor (AR), whereas DHT did not affect mRNA of the vitamin-D receptor (VDR). Coincubation with both steroid hormones did not stimulate receptor mRNAs more than either hormone alone. The proliferative effects of DHT and 1,25(OH)₂D₃ were completely inhibited by simultaneous incubation with both hormones, despite potentiation of IGF-I synthesis. In contrast, both hormones synergistically stimulated cell differentiation as judged by alkaline phosphatase activity, collagen X mRNA, and matrix calcification in long-term experiments. We conclude that DHT and 1,25(OH)₂D₃ interact with respect to chondrocyte proliferation and cell differentiation. The proliferative effects of both hormones are mediated by local IGF-I synthesis. Simultaneous coincubation with both hormones blunts the proliferative effect exerted by either hormone alone, in favor of a more marked stimulation of cell differentiation.     

Effect of 1,25-dihydroxyvitamin D3 on mouse mammary tumor (GR) cells: evidence for receptors, cellular uptake, inhibition of growth and alteration in morphology at physiologic concentrations of hormone

Mammary glands are target tissues for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We have examined a mouse mammary tumor cell line (GR) for receptors of 1,25(OH)2D3 and have examined alterations in the growth and morphology of these cells in response to 1,25(OH)2D3. GR cells contain a high affinity (Kd approximately 10(-11)), low-capacity receptor with a high specificity for 1,25(OH)2D3. The 1,25(OH)2D3 receptor in GR cells has a sedimentation coefficient of 3.5 and elutes from DEAE cellulose columns with approximately 0.15 M KCl. These properties of the receptor are similar to those reported for other 1,25(OH)2D3 receptors. 1,25(OH)2D3 is internalized by GR cells in situ and specifically bound 1,25(OH)2D3 is found predominantly, if not entirely, in the nucleus as determined by cell fractionation and autoradiographic techniques. The incubation of GR cells in culture for 7 days with 1,25(OH)2D3 markedly alters cell growth. Cell growth is retarded in a dose-dependent manner; physiologic concentrations (10(-10) M) of 1,25(OH)2D3 retard cell growth by approximately 50%. In addition, GR cells incubated with 10(-9) to 10(-8) M 1,25(OH)2D3 undergo marked morphological changes. The incubation of GR cells with other vitamin D metabolites such as 25-hydroxyvitamin D3 (25(OH)D3) at a concentration of 10(-9) M does not significantly alter cell growth or morphology. The presence of high affinity receptors for 1,25(OH)2D3, the specific internalization of 1,25(OH)2D3 predominantly into the nuclei, and the significant effects of physiological concentrations of 1,25(OH)2D3 on cell growth suggest a direct, specific, nuclear effect of 1,25(OH)2D3 on GR cells. The mouse mammary tumor model might be useful in examining the effect of 1,25(OH)2D3 on tumor formation.     

Effects of vitamin D metabolites on collagen production and cell proliferation of growth zone and resting zone cartilage cells in vitro

Previous studies have suggested that vitamin D metabolites directly influence the differentiation and maturation of chondrocytes in calcifying cartilage. Recently, this laboratory has shown that the response of chondrocyte plasma membrane and matrix vesicle enzymes to 1,25-(OH)2D3 and 24,25-(OH)2D3 is both cell and membrane specific. The current study demonstrates that cell replication and matrix protein synthesis are also modulated by vitamin D. Confluent, third-passage growth zone (GC) and resting zone (RC) costochondral chondrocytes were incubated in medium containing 10(-13)-10(-7) M 1,25-(OH)2D3 or 10(-12)-10(-6) M 24,25-(OH)2D3. The amount of collagenase-digestible protein (CDP) secreted into the media was inversely proportional to the concentration of fetal bovine serum (FBS). At 10% FBS, greater than 80% of the CDP was incorporated into the matrix. 1,25-(OH)2D3 stimulated CDP and percentage collagen synthesis by GC cells but had no effect on the synthesis of noncollagenous protein (NCP). 1,25-(OH)2D3 inhibited CDP and percentage collagen synthesis by RC cells but did not alter NCP synthesis. [3H]thymidine incorporation was inhibited in both cell types, whether confluent or subconfluent cultures were examined. At 10(-6) and 10(-7) M 24,25-(OH)2D3, there was a significant decrease in CDP production and percentage collagen synthesis by RC cells but no effect on NCP. However, at 10(-9) and 10(-10) M hormone there was an increase in NCP production but no effect on CDP, resulting in a decrease in percentage collagen synthesis. CDP and NCP production were unaffected by 24,25-(OH)2D3 in GC cells. High concentrations of hormone inhibited [3H]thymidine incorporation in both cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of endochondral bone formation: roles of growth hormone, 1,25-dihydroxyvitamin D and hyperparathyroidism

Impairment of linear growth occurs invariably in children with chronic renal failure. Recombinant human growth hormone and 1,25-dihydroxyvitamin D (calcitriol) are widely utilized to improve linear growth in children. Large doses of calcitriol, however, have been shown to suppress chondrocyte proliferation and may lead to the development of adynamic bone. Substantial reductions of growth have been shown in children with chronic renal failure treated with intermittent calcitriol therapy. These findings suggest that calcitriol can modify chondrocyte proliferation and/or differentiation in epiphyseal growth plate cartilage and may counteract the effects of growth hormone therapy in increasing linear growth in children with chronic renal failure. Parathyroid hormone related peptide (PTHrP) and its receptor (PTH/PTHrP receptor) play critical roles in regulating chondrocyte differentiation in the growth plate. The expression of PTH/PTHrP receptor mRNA is downregulated in animals with chronic renal failure and advanced secondary hyperparathyroidism; calcitriol and growth hormone therapy may modify the expression of PTH/PTHrP receptor. This article summarizes the separate and combined effects of growth hormone and calcitriol on endochondral bone formation in chronic renal failure and secondary hyperparathyroidism. Received: 10 September 1999 / Revised: 23 December 1999 / Accepted: 30 December 1999     

Expression of a 1,25-dihydroxyvitamin D3 membrane-associated rapid-response steroid binding protein during human tooth and bone development and biomineralization.

The calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been established to control skeletal tissue formation and biomineralization via the regulation of gene expression. This action involves the well-characterized nuclear 1,25(OH)2D3 receptor. However, it has been recognized that several cellular responses to 1,25(OH)2D3 may not to be related to the exclusive nuclear receptor. Indeed, this secosteroid is able to generate rapid responses that have been proposed to be mediated by interactions of the ligand, which is a putative cell membrane-associated rapid-response steroid (MARRS) binding protein for 1,25(OH)2D3 [1,25D3-MARRS]. The nongenomic pathway of 1,25(OH)2D3 was studied here in detail by immunolocalization of the 1,25D3-MARRS during the specific context of human prenatal development. Western blotting with proteins extracted from 4 week- to 27-week-old embryos was performed, evidencing a 65-kDa molecular species recognized by antibody Ab 099 generated against synthetic peptides corresponding to the N terminus of the 1,25D3-MARRS from chick intestinal basolateral membranes. Based on this biochemical conservation of protein in the human species, the temporospatial expression patterns were established in the craniofacial skeleton at the same ages. Comparative analysis was performed in teeth and bones from early morphogenesis to terminal cell differentiation and extracellular biomineralization. The data show the potential implication of 1,25D3-MARRS in the heterogeneous cell population including ameloblasts, odontoblasts, osteoblasts, and osteoclasts. The epithelial-mesenchymal cascade related to odontogenesis was coincident with a sequence of up- and down-regulation of immunoreactive 1,25D3-MARRS. Biomineralization was associated with a striking up-regulation in the adjoining secretory cells in all tissues. Finally, osteoclasts appeared also to express the 1,25D3-MARRS during these early phases of bone modeling. Previously obtained data of the nuclear vitamin D receptor (VDR) expression and this study on 1,25D3-MARRS suggest the existence of cross-talk between the genomic and nongenomic pathways during human development.     

Bone vitamin D-dependent calcium-binding protein is localized in chondrocytes of growth-plate cartilage

Summary The cellular distribution of vitamin D-dependent calcium-binding protein (CaBP) was examined in rat and chicken bone by immunocytochemical methods using an antiserum raised against purified chicken intestinal CaBP. In EDTA-decalcified, Vibratome sections of growing rat long bones, specific CaBP immunostaining was observed in cytoplasm of chondrocytes of the growth plate, particularly in regions of calcification. In undecalcified, frozen sections from neonatal rat, positive staining was seen in chondrocytes of tibial growth plate and also in chondrocytes of the long bones of the skull. No specific immunostaining was observed in osteoblasts, osteocytes or osteoclasts in mineralized bone. In frozen sections of tibias from 19-day-old chick embryos specific immunostaining was again confined to dividing chondrocytes of the growth plate and was much less intense in “resting” cartilage. The finding of CaBP in chondrocytes, cells known to possess specific receptors for 1,25-dihydroxyvitamin D₃ and to respond to the hormone, suggests a possible functional role for CaBP in chondrocyte maturation, differentiation and/or cartilage calcification.     

The influence of vitamin D metabolites on the calcification of cartilage matrix and the C-propeptide of type II collagen (chondrocalcin)

The influence of vitamin D metabolites (at 1 X 10(-10) M) on the calcification of cartilage matrix (measured by 45Ca2+ uptake) and the C-propeptide of type II collagen (measured by radioimmunoassay) has been studied using organ cultures and chondrocytes isolated from growth plates of vitamin D-deficient and -sufficient 11-day-old rats. Vitamin D-deficient rats had reduced amounts of C-propeptide in their serum and freshly isolated growth plate chondrocytes. In all chondrocytes cultured from vitamin D-deficient animals, the C-propeptide content was maximal at 24 hr whereas calcification continued to increase for up to 72 hr. In organ and chondrocyte cultures of tissue from vitamin D-sufficient rats, both 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25(OH)2D3) were required for maximal stimulation of calcification and maximal increases in C-propeptide content. In these D-replete tissues, 24,25-(OH)2D3 had a less stimulatory effect on both calcification (organ and cell cultures) and C-propeptide (organ cultures only), while 1,25(OH)2D3 alone had no effect in cell cultures but an inhibitory effect in organ cultures. Studies of cells or tissue from growth plates of vitamin D-deficient rats demonstrated that 24,25(OH)2D3 alone produced maximal calcification and maximal increases in the C-propeptide content. 1,25(OH)2D3 generally had an inhibitory effect on both calcification and C-propeptide when used alone. In the presence of 1,25(OH)2D3, the stimulatory effect of 24,25(OH)2D3 was partly abrogated. Maximal stimulation of calcification and increases in C-propeptide by 24,25(OH)2D3 were observed at 1 X 10(-9) M and 1 X 10(-10) M. In none of these studies was there any effect on proteoglycan content.(ABSTRACT TRUNCATED AT 250 WORDS)     

1alpha,25-dihydroxyvitamin D3 induced growth inhibition of PC-3 prostate cancer cells requires an active transforming growth factor beta signaling pathway

BACKGROUND: Prostate cancer growth inhibition by 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) is best characterized in the androgen dependent LNCaP cell line, where treatment with this hormone causes cell cycle arrest and apoptosis. 1,25(OH)2D3 also inhibits the growth of PC-3 prostate cancer cells, but not through the induction of G1 arrest or apoptosis. In this study, we have sought to elucidate the mechanism/s involved in PC-3 cell growth inhibition by 1,25(OH)2D3. EXPERIMENTAL METHODS: We determined the effect of transforming growth factor beta (TGFbeta) blocking antibodies on 1,25(OH)2D3 mediated growth inhibition of PC-3 cells. In addition, we also studied the effects of 1,25(OH)2D3 on TGFbeta signaling and receptor expression. Finally, we assessed the role of TGFbeta signaling in the induction of the growth inhibitory protein, insulin like growth factor binding protein 3 (IGFBP-3), by 1,25(OH)2D3. RESULTS: We find that 1,25(OH)2D3 action in PC-3 cells is mediated through at least two distinct pathways, the TGFbeta pathway and the IGFBP-3 pathway. We show that 1,25(OH)2D3 treatment elevates TGFbeta production and signaling, as well as receptor levels, in PC-3 cells. Further, using a blocking antibody against TGFbeta substantially reduces 1,25(OH)2D3 mediated growth inhibition without affecting IGFBP-3 induction, suggesting that IGFBP-3, alone, is insufficient to inhibit the growth of PC-3 cells.