1,25-dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)₂D₃ were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)₂D₃. In the present studies we have examined the ability of 1,25(OH)₂D₃ to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)₂D₃ increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)₂D₃ or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)₂D₃ were both rapid and transient. In addition, PKC_19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)₂D₃. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)₂D₃-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)₂D₃ following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)₂D₃ treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.     

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1α,25-(OH)₂D₃ (1,25) and 24R,25-(OH)₂D₃ (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1,25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKCα and ζ as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [γ³²P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A, (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25, but not 1,25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase. © 1996 Wiley-Liss, Inc.     

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.     

Intestinal Na/Ca exchanger protein and gene expression are regulated by 1,25(OH)2D3 in vitamin D-deficient chicks

The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.     

Quercetin exerts an inhibitory effect on cellular bioenergetics of the B164A5 murine melanoma cell line

This study examined the hypothesis that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) upregulates the insulin-independent signaling cascade of glucose metabolism. C2C12 myotubes were treated with high glucose (HG, 25 mM) and 1,25(OH)₂D₃ (0–50 nM). 1,25(OH)₂D₃ supplementation upregulated both insulin-independent (SIRT1) and insulin-dependent (p-IRS) signaling molecules, and stimulated the GLUT4 translocation, and glucose uptake in HG-treated myotubes. The effect of 1,25(OH)₂D₃ on IRS1 phosphorylation, GLUT4 translocation, and glucose uptake was attenuated in SIRT1-knockdown myotubes. Treatment with 1,25(OH)₂D₃, coupled with insulin, enhanced GLUT4 translocation and glucose uptake compared to treatment with either insulin or 1,25(OH)₂D₃ alone in HG-treated myotubes, which suggests that insulin-independent signaling molecules can contribute to the higher glucose metabolism observed in 1,25(OH)₂D₃ and insulin-treated cells. The data, therefore, suggest that 1,25(OH)₂D₃ increases glucose consumption by inducing SIRT1 activation, which in turn increases IRS1 phosphorylation and GLUT4 translocation in myotubes.     

Arachidonic acid is an autocoid mediator of the differential action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth plate chondrocytes

Prior studies have shown that 24,25-(OH)₂D₃ and 1,25-(OH)₂D₃ regulate protein kinase C (PKC) in costochondral chondrocytes in a cell maturation-dependent manner, with 1,25-(OH)₂D₃ affecting primarily growth zone (GC) cells and 24,25-(OH)₂D₃ affecting primarily resting zone (RC) cells. In addition, 1,25-(OH)₂D₃ has been shown to increase phospholipase A₂ activity in GC, while 24,25-(OH)₂D₃ has been shown to decrease phospholipase A₂ activity in RC. Stimulation of phospholipase A₂ in GC caused an increase in PKC, whereas inhibition of phospholipase A₂ activity in RC cultures increased both basal and 24,25-(OH)₂D₃-induced PKC activity, suggesting that phospholipase A₂ may play a central role in mediating the effects of the vitamin D metabolites on PKC. To test this hypothesis, RC and GC cells were cultured in the presence and absence of phospholipase A₂ inhibitors (quinacrine and oleyloxyethylphosphorylcholine [OEPC]), phospholipase A₂ activators (melittin and mastoparan), or arachidonic acid alone or in the presence of the target cell-specific vitamin D metabolite. PKC specific activity in the cell layer was determined as a function of time. Phospholipase A₂ inhibitors decreased both basal and 1,25-(OH)₂D₃-induced PKC activity in GC. When phospholipase A₂ activity was activated by inclusion of melittin or mastoparan in the cultures, basal PKC activity in RC was reduced, while that in GC was increased. Similarly, melittin and mastoparan decreased 24,25-(OH)₂D₃-induced PKC activity in RC and increased 1,25-(OH)₂D₃-induced PKC activity in GC. For both cell types, the addition of arachidonic acid to the culture media produced an effect on PKC activity that was similar to that observed when phospholipase A₂ activators were added to the cells. These results demonstrate that vitamin D metabolite-induced changes in phospholipase A₂ activity are directly related to changes in PKC activity. Similarly, exogenous arachidonic acid affects PKC in a manner consistent with activation of phospholipase A₂. These effects are cell maturation- and time-dependent and metabolite-specific. J. Cell. Physiol. 176:516–524, 1998. © 1998 Wiley-Liss, Inc.     

Protein kinase C isotypes in signal transduction for the 1,25D3-MARRS receptor (ERp57/PDIA3) in steroid hormone-stimulated phosphate uptake

We undertook studies to determine which isotype(s) of protein kinase C (PKC) is/are activated by ligand binding to the 1,25D₃-MARRS receptor (ERp57/PDIA3) and subsequent stimulation of phosphate uptake. Isolated intestinal epithelial cells from vitamin D-replete chicks were exposed to 1,25(OH)₂D₃ for 1, 3, or 5 min, thoroughly chilled, homogenized, and P₂ fractions (20,000 × g post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P₂ membranes 1 min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCα exhibited redistribution to membranes in a biphasic dose–response curve: slightly stimulated at the lowest dose, maximal at 300 pM 1,25(OH)₂D₃, and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCβ also revealed hormone-mediated differences, while those with anti PKCγ did not. RNAi studies were then performed with siRNA against PKCα or PKCβ. Untransfected cells treated with hormone for 7 min exhibited enhanced ³²P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased ³²P uptake in both controls (relative to untransfected controls), and hormone treated cells. However, control and transfected cells treated with hormone had equivalent levels of uptake. Western blot analyses confirmed decreased immunoreactivity in transfected cells. Chemical PKCα (safingol) and PKCβ ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] blockers also confirmed the results from siRNA and demonstrated decreased ³²P uptake in cells treated with 1,25(OH)₂D₃ plus blockers in comparison with cells treated with 1,25(OH)₂D₃ alone. Thus, PKCα and PKCβ are both involved in steroid-stimulated phosphate uptake.     

Phorbol myristate acetate transactivates the avian β3 integrin gene and induces αvβ3 integrin expression

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) transactivates the avian β₃ integrin gene whose promoter contains at least two vitamin D response elements, one of which is in close proximity to a candidate AP1 site (TGACTCA). Since fos/jun and steroid hormones interact to regulate gene expression, we asked whether phorbol-12-myristate-13-acetate (PMA), which stimulates binding of fos/jun to AP1 sites, transactivates the avian β₃ integrin gene and, if so, does the phorbol ester modulate 1,25(OH)₂D₃ induction of the gene. We find the candidate AP1 sequence comigrates with the consensus AP1 sequence on electromobility shift assay when incubated with recombinant c-jun protein. Furthermore, PMA prompts expression of β₃ integrin mRNA in the avian monocytic line, HD11. The increase in message reflects transactivation of the β₃ gene and is mirrored by plasma membrane appearance of the integrin heterodimer α_vβ₃. Moreover, attesting to the functional significance of PMA-enhanced α_vβ₃ expression, cells treated with concentrations of the phorbol ester that induce the β₃ gene, spread extensively on plastic, an event blocked by an anti-α_v antibody and a peptide mimetic known to inhibit α_vβ₃-mediated cell attachment. Interestingly, co-addition of 1.25(OH)₂D₃ and PMA prompts greater expression of α_vβ₃ than when the cells are exposed to either agent alone and PMA enhances 1,25(OH)₂D₃-induced β₃ integrin mRNA expression. Thus, PMA and 1,25(OH)₂D₃ impact on the avian β₃ integrin gene independently and in combination. © 1996 Wiley-Liss, Inc.     

A-ring analogues of 1,25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation

1,25-(OH)₂D₃ (1,25) and 24,25-(OH)₂D₃ (24,25) 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 analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10^-8 M 1,25, 10^-7 M 24,25, or the analogues at 10^-9–10^-6 M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH)₂D₃. Plasma membranes contained increased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)₂D₃. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)₂D₃, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)₂D₃ or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)₂D₃ as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Physiol. 171:357–367, 1997. © 1997 Wiley-Liss, Inc.     

Activation of rapid signaling pathways does not contribute to 1α,25‐dihydroxyvitamin D3‐induced growth inhibition of mouse prostate epithelial progenitor cells

The active form of vitamin D, 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D) inhibits the growth of prostate epithelial cells, however the underlying mechanisms have not been clearly delineated. In the current study, the impact of 1,25(OH)₂D on the rapid activation of extracellular‐regulated kinase (ERK) 1/2 and protein kinase C α (PKCα), and the role of these pathways in growth inhibition was examined in immortalized mouse prostate epithelial cells, MPEC3, that exhibit stem/progenitor cell characteristics. 1,25(OH)₂D treatment suppressed the growth of MPEC3 in a dose and time dependent manner (e.g., 21% reduction at three days with 100 nM 1,25(OH)₂D treatment). However, ERK1/2 activity was not altered by 100 nM 1,25(OH)₂D treatment for time points from 1 min to 1 h in either serum‐containing or serum‐free medium. Similarly, PKCα activation (translocation onto the plasma membrane) was not regulated by short‐term treatment of 100 nM 1,25(OH)₂D. In conclusion, 1,25(OH)₂D did not mediate rapid activation of ERK1/2 or PKCα in MPEC3 and therefore the growth inhibitory effect of 1,25(OH)₂D is independent of rapid activation of these signaling pathways in this cell type. J. Cell. Biochem. 107: 1031–1036, 2009. © 2009 Wiley‐Liss, Inc. This article was published online 2 June 2009. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 15 June 2009.     

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.     

Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D3

Retinoids and 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) induce differentiation of myeloid leukemia cells into granulocyte and macrophage lineages, respectively. All-trans retinoic acid (ATRA), which is effective in the treatment of acute promyelocytic leukemia, can induce differentiation of other types of myeloid leukemia cells, and combined treatment with retinoid and 1,25(OH)₂D₃ effectively enhances the differentiation of leukemia cells into macrophage-like cells. Recent work has classified macrophages into M1 and M2 types. In this study, we investigated the effect of combined treatment with retinoid and 1,25(OH)₂D₃ on differentiation of myeloid leukemia THP-1 and HL60 cells. 9-cis Retinoic acid (9cRA) plus 1,25(OH)₂D₃ inhibited proliferation of THP-1 and HL60 cells and increased myeloid differentiation markers including nitroblue tetrazolium reducing activity and expression of CD14 and CD11b. ATRA and the synthetic retinoic acid receptor agonist Am80 exhibited similar effects in combination with 1,25(OH)₂D₃ but less effectively than 9cRA, while the retinoid X receptor agonist HX630 was not effective. 9cRA plus 1,25(OH)₂D₃ effectively increased expression of M2 macrophage marker genes, such as CD163, ARG1 and IL10, increased surface CD163 expression, and induced interleukin-10 secretion in myeloid leukemia cells, while 9cRA alone had weaker effects on these phenotypes and 1,25(OH)₂D₃ was not effective. Taken together, our results demonstrate selective induction of M2 macrophage markers in human myeloid leukemia cells by combined treatment with 9cRA and 1,25(OH)₂D₃.     

1,25-dihydroxyvitamin D3 Activates MMP13 Gene Expression in Chondrocytes through p38 MARK Pathway

Osteoarthritis (OA) is the most prevalent degenerative joint disease. The highly regulated balance of matrix synthesis and degradation is disrupted in OA, leading to progressive breakdown of articular cartilage. The molecular events and pathways involved in chondrocyte disfunction of cartilage in OA are not fully understood. It is known that 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) is synthesized by macrophages derived from synovial fluid of patients with inflammatory arthritis. Vitmain D receptor is expressed in chondrocytes within osteoarthritic cartilage, suggesting a contributory role of 1,25-(OH)₂D₃ in the aberrant behavior of chondrocytes in OA. However, the physiological function of 1,25-(OH)₂D₃ on chondrocytes in OA remains obscure. Effect of 1,25-(OH)₂D₃ on gene expression in chondrocytes was investigated in this study. We found that 1,25-(OH)₂D₃ activated MMP13 expression in a dose-dependent and time-dependent manner, a major enzyme that targets cartilage for degradation. Interestingly, a specific mitogen-activated protein kinase p38 inhibitor SB203580, but not JNK kinase inhibitor SP600125, abrogated 1,25-(OH)2D3 activation of MMP13 expression. 1,25-(OH)₂D₃-induced increase in MMP13 protein level was in parallel with the phosphorylation of p38 in chondrocytes. To further address the effect of 1,25-(OH)₂D₃ on MMP13 expression, transfection assays were used to show that 1,25-(OH)₂D₃ activated the MMP13 promoter reporter expression. MMP13 is known to target type II collagen and aggrecan for degradation, two major components of cartilage matrix. We observed that the treatment of 1,25-(OH)₂D₃ in chondrocytes results in downregulation of both type II collagen and aggrecan while MMP13 was upregulated. Taken together, we provide the first evidence to demonstrate that 1,25-(OH)₂D₃ activates MMP13 expression through p38 pathway in chondrocytes. Since MMP13 plays a major role in cartilage degradation in OA, we speculate that the ability of 1,25-(OH)₂D₃ to potentiate MMP13 expression might facilitate cartilage erosion at the site of inflammatory arthritis.     

Modulation of myelomonocytic U937 cells by vitamin D metabolites

Investigation of the effects of 1,25(OH)₂D₃ and 24,25(OH)₂D₃ on the proliferation and differentiation of the human myelomonocytic cell line U937 has been complemented with studies of the effect of the same metabolites on the number of nuclear receptors for 1,25(OH)₂D₃. Both 1,25(OH)₂D₃ and 24,25(OH)₂D₃ inhibit the proliferation of U937 cells in a dose-dependent manner. The concentrations of 24,25(OH)₂D₃ required to produce this effect were 100-times greater than those of 1,25(OH)₂D₃. Inhibition of proliferation was associated with increased expression of the CD14 and 200 kDa 63D3 antigens thus confirming differentiation of U937 towards a more mature cell type.Studies of the nuclear receptor for 1,25(OH)₂D₃ showed that pre-treatment of the cells with 1,25(OH)₂D₃ resulted in an apparent 40% decrease in the number of detectable 1,25(OH)₂D₃ receptors as compared to control U937 cells. This is due to the fact that the 1,25(OH)₂D₃ binds to U937 cell nuclei during culture and thus blocks the subsequent binding of radiolabelled 1,25(OH)₂D₃ used to measure the number of 1,25(OH)₂D₃ receptors. Measurement of the binding of unlabelled 1,25(OH)₂D₃ by radioimmunoassay indicated that pre-treatment of the cells with 1,25(OH)₂D₃ increased the capacity of U937 to bind the hormone, although measurement of these receptors by whole cell assay was prevented by the binding of 1,25(OH)₂D₃ itself. This effect was not observed with 24,25(OH)₂D₃ which was more easily displaced from binding sites by radiolabelled 1,25(OH)₂D₃ and it appears to act through low affinity binding to the 1,25(OH)₂D₃ receptor.