A Vitamin D Analog Regulates Mesangial Cell Smooth Muscle Phenotypes in a Transforming Growth Factor-β Type II Receptor-mediated Manner*

Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-β (TGF-β) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D3 (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D3(1,25(OH)2D3) in a dose-dependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle α actin mRNA, but 1,25(OH)2D3-treated MC-E cells did not. The addition of a TGF-β1-neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels. However, after exposure to 1,25(OH)2D3 or OCT, there was no significant difference in the secretion of active TGF-β. We next investigated whether TGF-β type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.


Mesangial cells share features with contractile smooth muscle cells and mechanically support the capillary wall. The role of vitamin D compounds and the transforming growth factor-␤ (TGF-␤) type II receptor in modulating the smooth muscle phenotype of cultured mesangial cells was examined. Cell proliferation was significantly inhibited by the vitamin D analog 22-oxa-1,25-dihydroxyvitamin D 3 (22-oxacalcitriol; OCT) rather than by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) in a dosedependent manner. OCT-treated early passage mesangial cells (MC-E cells) had increased expression levels of type IV collagen and smooth muscle ␣ actin mRNA, but 1,25(OH) 2 D 3 -treated MC-E cells did not. The addition of a TGF-␤ 1 -neutralizing antibody to the OCT-treated MC-E cells blocked this inhibitory effect for cell proliferation and attenuated the up-regulated mRNA levels.
However, after exposure to 1,25(OH) 2 D 3 or OCT, there was no significant difference in the secretion of active TGF-␤. We next investigated whether TGF-␤ type II receptor (RII) was involved in this regulation. OCT treatment significantly increased the expression of the RII mRNA in MC-E cells. These results suggest that the vitamin D analog OCT induces smooth muscle phenotypic alterations and that this phenomenon was mediated through the induction of RII in cultured mesangial cells.
1,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) 1 was originally identified as a nutritional factor and a fat-soluble vitamin. Its physiological role is to regulate calcium metabolism. It was then reevaluated and came to be thought of as a kind of steroid hormone. The role of 1,25(OH) 2 D 3 has also been recognized as being much broader and thus much more physiologically important than originally thought, as reflected by its effects on the proliferation and differentiation of a variety of cells mainly including malignant and immune cells.
Since the first report (1) that 1,25(OH) 2 D 3 inhibited proliferation and induced the differentiation of murine myeloid leukemic M1 cells into monocyte macrophages, many studies have demonstrated that 1,25(OH) 2 D 3 is a potent modulator in suppressing proliferation and inducing the differentiation of numerous normal and tumor cells. However, the effective doses for these effects often cause hypercalcemia. To prevent this, many analogs of 1,25(OH) 2 D 3 have been synthesized. One of them, 22-oxa-1,25-dihydroxyvitamin D 3 (22-oxacalcitriol, OCT), has a more potent differentiation-inducing effect (2) than 1,25(OH) 2 D 3 without inducing hypercalcemia.
In the kidney, the anti-proliferative effects of 1,25(OH) 2 D 3 have been demonstrated in cultured mesangial cells (3). However, its differentiation-inducing effects have remained unknown. Mesangial cells (MCs) are uniquely differentiated cells, which acquire a smooth muscle-like phenotype, and are believed to be derived from mesenchymal cells (4). We have previously reported that cultured mesangial cells in the early passages (MC-E) (Ͻ25) showed a smooth muscle cell phenotype such as low cell turnover, high levels of expression for type IV collagen (Col IV) and smooth muscle ␣ actin (SMA), together with low expression of type I collagen (Col I). After multiple passages, mesangial cells in the late passages (MC-L) (Ͼ45) lost their smooth muscle phenotype and increased cell turnover (5).
In addition, evidence is accumulating that suggests that MCs, during the process of glomerular injuries (such as inflammation), can proliferate and undergo a phenotypic modulation in which they markedly up-regulate their expression of smooth muscle-like proteins (i.e. SMA). Furthermore, MCs can develop fibroblast-like characteristics by secreting interstitial collagens (i.e. type I and III collagens) that are not normally present in the mesangial matrix (6,7). However, there is little information regarding the mechanisms that underlie this phenotypic change.
TGF-␤ has widespread effects on extracellular matrix proliferation in many cultured cell lines and appears to play a role in the pathological accumulation of the extracellular matrix that accompanies inflammatory and fibrotic diseases such as glomerulonephritis (8). TGF-␤ may also have other important functions on glomerular epithelial and mesangial cells, including the regulation of cell proliferation, hypertrophy, and survival (apoptosis), as well as the modulation of the local and systemic immune response. Recent studies have shown that TGF-␤ overexpression in experimental and human kidney disease leads to progressive glomerular and tubulointerstitial scarring and renal failure (9 -11).
The loss of responsiveness to the inhibitory effects of TGF-␤ has been implicated in the development of a variety of cancers. The signal transduction of TGF-␤ is regulated via a heteromeric complex of type I and type II TGF-␤ receptors (RI and RII). An attenuation of TGF-␤ receptor expression is likely to play a critical role in the escape from this growth regulation.
Recent evidence indicates that a loss of RII is often associated with a failure to respond to autocrine and exogenous TGF-␤. In some cancer cell lines, RII expression is certainly a critical determinant for conferring TGF-␤ tumor suppression as well as negative autocrine TGF-␤ growth functions. A study by Wu et al. (12) in human breast cancer cells demonstrated that increased RII mRNA and protein levels following treatment with 1,25(OH) 2 D 3 and its analogs contributed to TGF-␤ activity. It is generally accepted that 1,25(OH) 2 D 3 works in context with TGF-␤ in controlling cell proliferation. However, with respect to cell differentiation, it is still undetermined how 1,25(OH) 2 D 3 and TGF-␤ interact. In this study, we investigated the mechanisms underlying the differentiation of the MC phenotype in vitro.

EXPERIMENTAL PROCEDURES
Vitamin D 3 Compounds-1,25(OH) 2 D 3 was purchased from Philips Duphar Co. (Amsterdam, The Netherlands), and its synthetic analog OCT was generously supplied by Chugai Pharmaceutical Co. Ltd. (Tokyo, Japan). Stock solutions were prepared in absolute ethanol at 10 Ϫ4 M. Serial dilutions were then made in absolute ethanol and stored at Ϫ20°C protected from light. These diluted solutions were added to the experimental culture medium at a final ethanol concentration of 1%. The control cells received 1% ethanol vehicle, which had no effect on cell proliferation (data not shown).
Cell Culture-A glomerular mesangial cell line was established from glomeruli isolated from normal, 4-week-old mice (C57BL/6JxSJL/J) and was identified according to the method previously described (13,14). The mesangial cells were plated on 100-mm plastic dishes (Nunc) and maintained in B medium (a 3:1 mixture of minimal essential medium/ F12 modified with trace elements) supplemented with 1 mM glutamine, penicillin at 100 units/ml, streptomycin at 100 mg/ml, and 20% fetal calf serum (Irvine Scientific). The cells were passaged weekly with trypsin-EDTA. The cultured cells fulfilled the criteria generally accepted for glomerular MCs previously described (4,15).
Cell Proliferation-[ 3 H]Thymidine incorporation was performed to assess the DNA synthesis rate. The mesangial cells were plated at 2.5 ϫ 10 4 cells/well in 24-well plates for 24 h, and the medium was replaced with serum-free medium containing 0.1% bovine serum albumin and the incubation continued for another 24 h. Various concentrations of compounds (1, 25(OH) 2 D 3 , OCT, or vehicle) and [ 3 H]thymidine (1 m Ci/ml, 1 Ci/well, NEN Life Science Products) were then added, and the cells were incubated for 24 h in 2.0% fetal calf serum. The cells were washed, precipitated with 10% trichloroacetic acid, and counted in a liquid scintillation counter (16). The experiments were performed in quadruplicate.
RNase Protection Assay-Total RNA was isolated from mesangial cells using the TRIzol reagent (Life Technologies, Inc.) and was analyzed by a RNase protection assay with the following probes. The RNA probes were prepared by linearizing the PvuII fragment of Col IV (␣1) from p1234, the HindIII fragment of SMA from pSA2HE, the ApaI fragment of Col I (␣2) from pGM101, and the EcoRI fragment of GAPDH from pMGAP1 (17)(18)(19). In addition, we constructed a RII riboprobe to measure the RII mRNA levels. A fragment of mouse RII cDNA (196 base pairs) was obtained by polymerase chain reaction using the primer pair 5Ј-GCAAGTTTTGCGATGTGAGA-3Ј and 5Ј-GCATCT-TCCAGAGTGAAGCC-3Ј, according to the sequence published by Suzuki et al. (20). The polymerase chain reaction fragment was confirmed to be the RII cDNA by DNA sequencing and was cloned into a pCR II-TOPO plasmid (Invitrogen). After digesting the plasmid with SacI, an antisense riboprobe was synthesized in vitro using T7 RNA polymerase, which protects a 196-base pair fragment. The RNA probe (4 ϫ 10 5 cpm) and the test RNA were hybridized overnight at 45°C. RNase A (40 g/ml) and RNase T1 (2 g/ml) were added to each tube, and the tubes were incubated for 1 h at 30°C. The RNase resistant fragments were analyzed by 3.5 or 7% polyacrylamide-8 M urea gel electrophoresis and autoradiography (5,21). The protected bands for each RNA probe had the same size as the coding sequence for the specific mRNA, thus providing evidence for their specificity, and were evaluated by densitometric analysis.
TGF-␤-neutralizing Antibody Assay-The cells were resuspended at a concentration of 5 ϫ 10 4 cells/ml and plated onto 24-well plates (500 l/well) in the presence of either 10 g/ml TGF-␤1 neutralizing antibody (R&D Systems) or a control normal IgG. After 3 h of incubation different concentrations of vitamin D 3 compounds were added as indi-cated. The cells were allowed to grow for 72 h without changing the medium followed by the determination of the [ 3 H]thymidine incorporation as described above.
Mink Lung Epithelial Cell Growth Inhibition Assay-The cells were plated onto 100-mm dishes and allowed to reach 70 -80% confluency. The medium was removed and replaced with 5 ml of fresh B medium. The cells were then treated with 1,25(OH) 2 D 3 , OCT, or vehicle only for 24 h. Following this treatment, the conditioned medium was collected, and the indicated volumes were used to treat mink lung epithelial cells plated on 96-well plates at a density of 1500 cells/well. In addition, a standard TGF-␤ growth inhibition curve was generated by treating the cells with various concentrations of TGF-␤1. The mink lung epithelial cells were allowed to incubate for 3 days, at which time the medium was removed and replaced by 100 l of fresh medium. The colonies were immediately visualized by staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) for 2 h. The stained cells were solubilized with Me 2 SO (Sigma), and the relative cell numbers were determined by the resultant absorbance at 595 nm.
DNA Transfection-The Col IV (␣1) promoter/enhancer/chloramphenicol acetyltransferase (CAT) construct (p56) has been previously described (22). The construct SMP-1 contains 1074 base pairs of the proximal 5Ј-flanking region plus 43 base pairs of the 5Ј-untranslated region of the SMA gene cloned into the SalI-BamHI site of the SV40 enhancer containing CAT vector pBLCAT3 (23). Next, 4 ϫ 10 5 cells were plated on a 60-mm dish and transfected with plasmid DNA using the LipofectAMINE PLUS Reagent (Life Technologies, Inc.). For each transfection, 2 g of CAT reporter plasmid and 0.5 g of pSV-␤-galactosidase (Promega) (internal control for transfection efficiency) were used. Forty eight hours after the start of the transfection the cells were harvested and assayed for CAT and ␤-galactosidase activity (24,25). CAT (Amersham Pharmacia Biotech) or ␤-galactosidase (Promega) was used as the standard, and pSV0CAT and pSV2CAT were used for negative (0%) and positive (100%) controls, respectively (26). A correction factor for differences in the transfection efficiency was obtained by normalizing the CAT activity to the ␤-galactosidase activity and then subtracting the background level, as determined by transfection with pSV0CAT. The final value was expressed as the relative CAT activity.
Statistical Analysis-Data were expressed as mean Ϯ S.E. and analyzed by an unpaired Student's t test.

RESULTS
Vitamin D 3 Sensitivity-The effects of 1,25-dihydroxyvitamin D 3 and its analog on the cell proliferation of MC-E and MC-L cells were investigated by assessing the [ 3 H]thymidine incorporation following treatment by these compounds. Both 1,25(OH) 2 D 3 and OCT inhibited DNA incorporation of the MC-E cells in a dose-dependent manner. The effect of OCT is more prominent than that of 1,25(OH) 2 D 3 (Fig. 1A). In contrast, these compounds did not significantly affect the MC-L cells (Fig. 1B).
Expression of SMA, Col IV, and Col I mRNA-The mRNAs for SMA, Col IV, and GAPDH were easily detectable in the untreated MC-E cells ( Fig. 2A), whereas Col I mRNA was poorly detected. After exposure to 1,25(OH) 2 D 3 or OCT, the OCT treatment showed an approximately 1.5-and 4-fold induction of SMA and Col IV mRNA respectively, whereas the expression levels of Col I remained unchanged. 1,25(OH) 2 D 3 treatment did not result in any change. In contrast, treatment with these compounds showed no change in the SMA and Col IV mRNA expression of the MC-L cells (Fig. 2B); however, the Col I mRNA levels were increased 2.2-fold in the OCT-treated MC-L cells. The GAPDH mRNA levels were equivalent in all cells.
Transcriptional Activity of SMA and Col IV-Transcriptional activity was investigated by transiently transfecting cells with fusion constructs containing a promoter/enhancer/ CAT reporter for each gene. The OCT-treated cells or control cells were transfected with the target constructs. The transcriptional activity of the SMA gene promoter was analyzed by the construct SMP-1, which contained the SMA promoter, the SV40 enhancer, and the CAT reporter gene. The CAT activity was increased 3.7-fold in the OCT-treated cells as compared with the control cells, demonstrating that OCT enhanced the transcriptional activity of the SMA gene. A p56 construct was used to evaluate the transcriptional activity of Col IV. OCT induced a 9.4-fold increase in CAT activity as compared with the control, thus indicating that OCT significantly stimulated the promoter activity of the Col IV (␣1) gene (Fig. 3). These data suggest that increased mRNA levels for SMA and Col IV (␣1) induced by OCT were because of increased promoter activity in their genes.
TGF-␤ Autocrine Activity-To elucidate the correlation between TGF-␤ and vitamin D 3 sensitivity, a TGF-␤-neutralizing antibody assay was performed. At 10 g/ml, the TGF-␤-neutralizing antibody reversed the inhibitory effects of vitamin D 3 and its analog OCT; the antibody generated an approximately 50% increase in DNA synthesis as compared with normal chicken IgG treatment, in a dose-dependent manner, on MC-E cells (Fig. 4A). In contrast, the MC-L cells did not respond to the TGF-␤-neutralizing antibody (Fig. 4B), thus indicating a lack of induction of autocrine TGF-␤ activity. These results indicate that the proliferation inhibitory mechanisms of vitamin D 3 involve the induction of TGF-␤ activity in MC-E cells. Moreover, the addition of TGF-␤-neutralizing antibody attenuated the up-regulated mRNA levels (data not shown).
Active TGF-␤ Levels in MCs Treated with Conditioned Me-dium-The increased autocrine TGF-␤ activity could have resulted from the enhanced expression of TGF-␤ and/or its receptors. To test these possibilities, an inhibition bioassay on mink lung epithelial cells was performed. As a standard, the addition of TGF-␤1 inhibited the proliferation of MC-E cells in a dosedependent fashion (data not shown). Conditioned medium from 1,25(OH) 2 D 3 (10 Ϫ6 M) or OCT (10 Ϫ6 M)-treated and -untreated MC-E cells was added to mink lung epithelial cells. After exposure to either treated or untreated conditioned medium, no significant differences in proliferation inhibition were observed in the mink lung epithelial cells (Fig. 5). Similarly, no significant differences in proliferation inhibitory effects were observed in the MC-L cells (data not shown). Moreover, the other conditioned medium, 10 Ϫ10 or 10 Ϫ8 M 1,25(OH) 2 D 3 or OCT, resulted in no significant difference. These results demonstrated that vitamin D 3 compounds did not alter the activation of secreted growth and/or inhibitory cytokines from MC-E cells, one of which is likely to be TGF-␤1. Furthermore, these results also suggest that the enhanced TGF-␤ autocrine activity by the vitamin D 3 compounds did not result from the modulation of ligand activation.
Expression of RII mRNA Levels-The other possibility for the increased autocrine TGF-␤ activity following treatment with vitamin D 3 compounds was the induction of receptor expression; therefore, we determined whether 1,25(OH) 2 D 3 and its analog could modulate the expression of RII mRNA. 1,25(OH) 2 D 3 (10 Ϫ6 M) or OCT (10 Ϫ6 M) were utilized to determine the kinetic effects on RII expression. MC-E cells untreated with vitamin D 3 compounds expressed RII mRNA 6-fold higher than MC-L cells. After exposure to 1,25(OH) 2 D 3 and OCT, a 2.1-and 4.5-fold increase, respectively, in RII mRNA expression levels in MC-E cells was observed. On the other hand, OCT-treated MC-L cells showed a 1.8-fold increase in their RII mRNA levels, but no significant modulation was noted in 1,25(OH) 2 D 3 -treated MC-L cells (Fig. 6). DISCUSSION MCs possess a specific smooth muscle phenotype including contractile properties, an extensive array of microfilaments, and the production of basement components. In several studies, cultured MCs exhibited an increasingly dedifferentiated phe-notype as they were passaged (27,28). These dedifferentiated MCs can produce interstitial collagen and gain the capability to proliferate. In this study, we first demonstrated that a vitamin D 3 analog could induce a smooth muscle phenotype in cultured MCs. The induction of smooth muscle phenotype could be related to TGF-␤ signaling. However, active TGF-␤ levels were not enhanced by the vitamin D 3 compounds in this study. The increased cellular responsiveness to TGF-␤ was correlated with a significant induction of RII expression by the vitamin D 3 analog. This study suggests that the vitamin D 3 analog may be a new modulator for the smooth muscle phenotype and that the mechanisms underlying this process are tightly related to the regulation of RII expression.
The signal transduction of TGF-␤ is mediated via two different types of serine/threonine kinase receptors, RI and RII. RII is the primary binding protein for ligands and is the most important for signal transduction. RII transactivates RI, which transduces various signals via the Smad family (29 -31). Next, we examined the levels of RII mRNA expression. The low expression of RII mRNA in the MC-L cells supports the connection that there was no inhibition of cell proliferation because of the vitamin D 3 compounds. Increased RII mRNA expression was observed after treatment with OCT rather than  1 and 4) in the MC-E (lanes 1-3) and MC-L cells (lanes 4 -6) for 24 h, the specific mRNAs were determined by using the RNase protection assay. The amount of total RNA loaded (1 g/lane) was checked by hybridization with a GAPDH probe. The data are representative of two independent experiments. 1,25(OH) 2 D 3 in the MC-E cells, corresponding to the higher ability of OCT to inhibit cell proliferation. The TGF-␤-neutralizing antibody prevented inhibitory effects of cell proliferation by vitamin D 3 compounds. These effects seemed to be correlated with the increasing amounts in RII expression induced by 1,25(OH) 2 D 3 or OCT. Therefore, these data suggest that TGF-␤ signal transduction significantly prevents these effects of vitamin D 3 compounds. Moreover, the stimulatory and inhibitory Smad families may alter the feedback after OCT treatment.
Thus, 1,25(OH) 2 D 3 and its analog can inhibit cell proliferation through the induction of negative autocrine TGF-␤ activity. However, the effective dose of 1,25(OH) 2 D 3 , which could induce negative autocrine TGF-␤ activity would also cause hypercalcemia, thus leading to unwanted adverse effects. To overcome this problem, 1,25(OH) 2 D 3 analogs such as OCT have been developed that have increased potency and a reduced hypercalcemic effect (32). We have demonstrated here that the analog OCT has similar effects as 1,25(OH) 2 D 3 at lower concentrations, which make it an attractive and potential antiproliferative agent against proliferative glomerulonephritis. The marked induction of differentiation caused by OCT was similar to the phenomena observed in leukemia cells, T cells, breast cancer cells, fibroblasts, and keratinocytes (2,(33)(34)(35). To elucidate the mechanisms responsible for the progression of glomerular injuries, it is indispensable to appreciate the phenotypic changes as well as the proliferation of mesangial cells. TGF-␤ has been implicated in the differentiation of vascular smooth muscle cells and other mesenchyme-derived cells during development (36). For example, the TGF-␤ treatment of human smooth muscle cells (37), granulation tissue myofibroblasts (38), and pericytes (39) has been shown to increase the expression of SMA, a marker of differentiated smooth muscle cells (40). However, the molecular basis for this phenotypic modulation and differentiation in cultured MCs is as yet poorly understood. Furthermore, OCT may offer an advantage in that the induction of the autocrine negative TGF-␤ activity occurs through RII and not the TGF-␤ ligand and thus could avoid damaging adjacent tissues.
A dedifferentiated smooth muscle phenotype can be observed in cultured MCs after multiple passages. These dedifferentiated cells have decreased RII expression and have lost their responsiveness to TGF-␤, which may be related to the dedifferentiation process. On the other hand, RII can be detected on MCs in vivo according to our recent observations. These findings suggest that RII could be a key regulator of MC differentiation and that OCT is an important modulator for RII expression. Further work is in progress to clarify the role of RII in the differentiation mechanisms of MCs in vivo and in vitro.