UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol delta7-reductase inhibitor

The skin fulfills an important role in the vitamin D photo-endocrine system. Epidermis is not only the site of vitamin D3 photoproduction. In addition, epidermal keratinocytes contain the vitamin D receptor (VDR) and possess 25-hydroxylase and 1alpha-hydroxylase activity indicating that all components of the vitamin D system are present. We investigated whether these components cooperate in inducing vitamin D activity upon treatment with physiological UVB doses. Upon irradiation, 24-hydroxylase mRNA was induced in keratinocytes pretreated with a sterol Delta7-reductase inhibitor (BM15766) whereby the 7-dehydrocholesterol content increased by 300-fold. Transfection experiments with a vitamin D response element containing construct confirmed VDR-dependent gene activation. Furthermore, the UVB-dependent induction of 24-hydroxylase was blocked by the cytochrome-P450 inhibitor ketoconazole. The 24-hydroxylase inducing photoproduct was transferable to unirradiated keratinocytes by medium and cellular homogenates of UVB-irradiated, BM15766-pretreated cells and was identified as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by high-performance liquid chromatography with tandem mass spectrometric detection. Addition of vitamin D binding protein blunted UVB-induced 24-hydroxylase suggesting the possibility of a paracrine or autocrine role for 1,25(OH)2D3. In conclusion, epidermal keratinocytes can produce vitamin D3, convert it to 1,25(OH)2D3 and respond to it upon UVB irradiation in the absence of exogenous 7-dehydrocholesterol and therefore contain a unique and complete photo-endocrine vitamin D system.     

Molecular pathways involved in the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 in primary human keratinocytes

We previously reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] protects primary human keratinocytes against ultraviolet (UV)B-induced apoptosis. Here, we confirmed the anti-apoptotic effect of 1,25(OH)2D3 in keratinocytes, using cisplatin and doxorubicin as apoptotic triggers. We further showed that 1,25(OH)2D3 activates two survival pathways in keratinocytes: the MEK/extracellular signal regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. Activation of ERK and Akt by 1,25(OH)2D3 was transient, required a minimal dose of 10(-9) M and could be blocked by actinomycin D and cycloheximide. Moreover, inhibition of Akt or ERK activity with respectively a PI-3K inhibitor (LY294002) or MEK inhibitors (PD98059, UO126), partially or totally suppressed the anti-apoptotic capacity of 1,25(OH)2D3. Finally, 1,25(OH)2D3 changed the expression of different apoptosis regulators belonging to the Bcl-2 family. Indeed, 1,25(OH)2D3 treatment increased levels of the anti-apoptotic protein Bcl-2 and decreased levels of the pro-apoptotic proteins Bax and Bad in a time- and dose-dependent way. Induction of Bcl-2 by 1,25(OH)2D3 was further shown to be mediated by ERK and, to a lesser extent, by Akt. In conclusion, 1,25(OH)2D3 clearly protects keratinocytes against apoptosis (1) by activating the MEK/ERK and the PI-3K/Akt survival pathways and (2) by increasing the Bcl-2 to Bax and Bad ratio.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

1,25-dihydroxyvitamin D(3) stimulated protein kinase C phosphorylation of type VI adenylyl cyclase inhibits parathyroid hormone signal transduction in rat osteoblastic UMR 106-01 cells

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) treatment of osteoblastic cells was shown previously to attenuate Parathyroid hormone (PTH) response by inhibiting adenylyl cyclase (AC) activity. In this study, we have investigated the mechanism by which 1,25(OH)(2)D(3) inhibits AC in rat osteoblastic UMR 106-01 cells. 1,25(OH)(2)D(3) treatment inhibited both PTH and forskolin-stimulated AC activity by 25%-50% within 12 min in a concentration-dependent manner suggesting a direct inhibition of the AC enzyme. Treatment with 25(OH)D(3) had no effect on basal or stimulated AC activity. We determined the profile of AC subtypes expressed in UMR cells and found AC VI to be the dominant subtype accounting for 50% of AC mRNA. Since AC VI can be inhibited by protein kinase C (PKC) phosphorylation, we examined 1,25(OH)(2)D(3) activation of various PKC isoforms. 1,25(OH)(2)D(3) increased the membrane translocation of PKC-betaI, -delta, and -zeta with a concomitant increase in PKC activity. The translocation of PKC-betaI and -delta was blocked by the PLC inhibitor U73122 whereas that of PKC-zeta was abolished by the PI-3 kinase inhibitor wortmannin. The attenuation of cAMP production by 1,25(OH)(2)D(3) was antagonized by the PKC inhibitors Go6850, calphostin C, and wortmannin, but not by a calmodulin kinase II (CaMKII) inhibitor. Treatment with 1,25(OH)(2)D(3) for 20 min increased AC VI phosphorylation by 10.8-fold and this was blocked partially by Go6850 and partially by wortmannin but was unaffected by CaMKII inhibitor. These results demonstrate that 1,25(OH)(2)D(3) activation of PKC isoforms leads to phosphorylation of AC VI and inhibition of PTH-activation of this pathway in osteoblasts.     

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.     

The MRNA expression of the human 1,25-dihydroxyvitamin D3 receptor and the c-myc protooncogene in cultured human keratinocytes

Summary The human vitamin D receptor mRNA expression in preconfluent human cultured keratinocytes was upregulated by treatment of these cells with 10^−8 M 1,25(OH)₂D₃ for 24 hours. Additionally, human c-myc mRNA expression was decreased in a dose dependent manner by 1,25(OH)₂D₃ in both preconfluent and confluent cultured human keratinocytes.     

Critical role of ASK1 in the 6-hydroxydopamine-induced apoptosis in human neuroblastoma SH-SY5Y cells

6-hydroxydopamine (6-OHDA)-induced apoptosis in dopaminergic neuronal cells is a common cell model of Parkinson's disease (PD). The role of apoptosis signal-regulating kinase 1 (ASK1) in this model has not been well studied. We observed significant activation of ASK1, p38 and JNK, as well as apoptosis in human dopaminergic neuroblastoma SH-SY5Y cells exposed to 6-OHDA. Over-expressing kinase-dead mutant ASK1(K709M) or knock-down of endogenous ASK1 by its small interfering RNA (siRNA) greatly suppressed activation of these kinases and apoptosis in the cells. It was found that the activation of p38 and JNK was suppressed to almost the same extent as that of ASK1 in the ASK1-knock-down cells, suggesting that activated ASK1 is almost totally responsible for activation of p38/JNK. It was also observed that the 6-OHDA-induced cell apoptosis could be effectively prevented by over-expressing the dominant-negative mutant of p38 or p38 inhibitor SB203580, demonstrating that activation of p38/JNK signalling is required for initiating the programmed cell death. Furthermore, suppression of the 6-OHDA-generated reactive oxygen species (ROS) by pre-incubation of cells with N-acetyl-L-cysteine effectively inhibited the 6-OHDA-induced activation of ASK1, p38 and JNK, and protected the cells from apoptosis. This study clearly shows the route from ROS generation by 6-OHDA to initiation of p38/JNK signalling via activation of ASK1 in the studied PD model.     

Ursolic acid overcomes Bcl‐2‐mediated resistance to apoptosis in prostate cancer cells involving activation of JNK‐induced Bcl‐2 phosphorylation and degradation

Androgen‐independent prostate cancers express high levels of Bcl‐2, and this over‐expression of Bcl‐2 protects prostate cancer cells from undergoing apoptosis. Ursolic acid (UA) has demonstrated an anti‐proliferative effect in various tumor types. The aim of this study is to evaluate the difference between UA‐induced apoptosis in androgen‐dependent prostate cancer cell line LNCaP cells and androgen‐independent prostate cancer cell line LNCaP‐AI cells and to reveal the molecular mechanisms underlying the apoptosis. We found that UA treatment in vitro can effectively induce apoptosis in LNCaP and LNCaP‐AI cells. UA can overcome Bcl‐2‐mediated resistance to apoptosis in LNCaP‐AI cells. Intrinsic apoptotic pathways can be triggered by UA treatment because c‐Jun N‐terminal kinase (JNK) is activated and subsequently provokes Bcl‐2 phosphorylation and degradation, inducing activation of caspase‐9. Although further evaluation is clearly needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. J. Cell. Biochem. 109: 764–773, 2010. © 2009 Wiley‐Liss, Inc.     

Compound K protects MIN6N8 pancreatic β‐cells against palmitate‐induced apoptosis through modulating SAPK/JNK activation

Chronic elevation of NEFAs (non‐esterified fatty acids) due to insulin resistance and obesity has been shown to be associated with increased β‐cell apoptosis and with the aetiology of the reduced β‐cell mass of Type 2 diabetes. SAPK (stress‐activated protein kinase)/JNK (c‐Jun N‐terminal kinase) have been implicated in the control of apoptosis. C‐K [compound K; 20‐O‐β‐d ‐glucopyranosyl‐20(S)‐protopanaxadiol] is the main intestinal bacterial metabolite of protopanaxadiol ginsenosides. Currently, little is known about the effects of C‐K on β‐cells with the presence of NEFAs. The aim of the present study was to investigate the in vitro protective effect of C‐K on MIN6N8 mouse insulinoma β‐cells against NEFA‐induced apoptosis, as well as the modulating effect on SAPK/JNK activation. Our results have shown that C‐K inhibited the palmitate‐induced apoptosis through modulating SAPK/JNK activation. We conclude that C‐K protects against β‐cell death and that, by anti‐apoptotic activity, C‐K may contribute to the previously reported anti‐diabetic actions of ginseng.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Stimulatory effect of 1,25-dihydroxyvitamin D3 on the glucose-6-phosphate dehydrogenase activity in the MCF-7 human breast cancer cell line

Human breast cancer cell lines have been shown to possess high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and their growth is inhibited by this steroid. The present study examines the effect of 1,25(OH)2D3 on the activity of glucose-6-phosphate dehydrogenase (G6PD) in cells of a human breast cancer cell line MCF-7. G6PD, an enzyme which controls the hexose monophosphate shunt, is elevated and sensitive to 17 beta-estradiol in breast tumors. G6PD activity was stimulated by 1,25(OH)2D3 in a dose-dependent manner at very low concentrations of steroid (10(-10)-10(-12) M). 1,25(OH)2D3 increased maximum velocity without modifying the affinity constant of the enzyme for glucose-6-phosphate.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Tiludronate inhibits interleukin-6 synthesis in osteoblasts: Inhibition of phospholipase D activation in MC3T3-E1 cells

In previous studies, we have reported that PGF_2α stimulates phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein in osteoblast-like MC3T3-E1 cells, and that PGF_2α and PGE₁ induce interleukin-6 (IL-6) synthesis via activation of protein kinase C and protein kinase A, respectively. In the present study, we investigated the effect of tiludronate, a bisphosphonate known to inhibit bone resorption, on the PGF_2α- and PGE₁-induced IL-6 synthesis in these cells. Tiludronate significantly suppressed the PGF_2α-induced IL-6 secretion in a dose-dependent manner in the range between 0.1 and 30 μM. However, the IL-6 secretion induced by PGE₁ or (Bu)₂cAMP was hardly affected by tiludronate. The choline formation induced by PGF_2α was reduced by tiludronate dose-dependently in the range between 0.1 and 30 μM. On the contrary, tiludronate had no effect on PGF_2α-induced formation of inositol phosphates. Tiludronate suppressed the choline formation induced by NaF, known as an activator of heterotrimeric GTP-binding protein. However, tiludronate had little effect on the formation of choline induced by TPA, a protein kinase C activator. Tiludronate significantly inhibited the NaF-induced IL-6 secretion in human osteoblastic osteosarcoma Saos-2 cells. These results strongly suggest that tiludronate inhibits PGF_2α-induced IL-6 synthesis via suppression of phosphatidylcholine-hydrolyzing phospholipase D activation in osteoblasts, and that the inhibitory effect is exerted at the point between heterotrimeric GTP-binding protein and phospholipase D. J. Cell. Biochem. 69:252–259, 1998. © 1998 Wiley-Liss, Inc.     

1alpha,25-Dihydroxyvitamin D3-3beta-(2)-bromoacetate,an affinity labeling derivative of 1alpha,25-dihydroxyvitamin D3 displays strong antiproliferative and cytotoxic behavior in prostate cancer cells

In this report we describe that 1,25(OH)(2)D(3)-3-BE, a VDR-affinity labeling analog of 1,25(OH)(2)D(3), showed strong and dose-dependent growth-inhibitory effect in several epithelial cells, i.e., keratinocytes (primary cells), MCF-7 breast cancer, PC-3, and LNCaP prostate cancer and PZ-HPV-7 immortalized normal prostate cell-lines. Furthermore, 10(-6) M of 1,25(OH)(2)D(3)-3-BE induced apoptosis specifically in LNCaP and PC-3 cells; and the effect was much less pronounced at lower doses. We also showed that the effect (of 1,25(OH)(2)D(3)-3-BE) was not due to probable degradation (hydrolysis) of 1,25(OH)(2)D(3)-3-BE or random interaction of this molecule with cellular proteins. Tissue- or cell-specific action of 1,25(OH)(2)D(3) and its mimics is not common due to the ubiquitous nature of VDR. Furthermore, variable effects of 1,25(OH)(2)D(3) and its analogs in various cell-lines potentially limits their application as anticancer agents. We showed that 1,25(OH)(2)D(3)-3-BE displayed similar growth-inhibitory and cytotoxic activities towards androgen sensitive LNCaP and androgen-independent PC-3 cell-lines. Therefore, these results raise the possibility that 1,25(OH)(2)D(3)-3-BE or similar VDR-cross linking analogs of 1,25(OH)(2)D(3) might be considered for further development as potential candidates for prostate cancer.     

1,25-Dihydroxyvitamin D3 induces Ca-mediated apoptosis in adipocytes via activation of calpain and caspase-12

Induction of apoptotic cell death is emerging as a promising strategy for prevention and treatment of obesity because removing of adipocytes via apoptosis may result in reducing body fat and a long-lasting maintenance of weight loss. However, the mechanisms controlling adipocyte apoptosis are unknown and even the ability of adipocytes to undergo apoptosis has not been conclusively demonstrated. We have shown previously that the specific Ca²⁺ signal, sustained increase in intracellular Ca²⁺, triggers apoptotic cell death via activation of Ca²⁺-dependent proteases and that the apoptosis-inducing effect of the hormone 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is mediated through Ca²⁺ signaling. Here, we report that 1,25(OH)₂D₃ induces apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca²⁺-dependent calpain and Ca²⁺/calpain-dependent caspase-12. Treatment of adipocytes with 1,25(OH)₂D₃ induced, in concentration- and time-dependent fashion, a sustained increase in the basal level of intracellular Ca²⁺. The increase in Ca²⁺ was associated with induction of apoptosis and activation of μ-calpain and caspase-12. Our results demonstrate that Ca²⁺-mediated apoptosis can be induced in mature adipocytes and that the apoptotic molecular targets activated by 1,25(OH)₂D₃ in these cells are Ca²⁺-dependent calpain and caspase-12. These findings provide rationale for evaluating the role of vitamin D in prevention and treatment of obesity.     

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)₂D₃ which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)₂D₃. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)₂D₃, 24,25-(OH)₂D₃, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)₂D₃ and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)₂D₃ having a greater effect. 24,25-(OH)₂D₃ caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)₂D₃, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation–dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457–470, 1997. © 1997 Wiley-Liss, Inc.