1,25-Dihydroxyvitamin D3 and 20-Hydroxyvitamin D3 Upregulate LAIR-1 and Attenuate Collagen Induced Arthritis.

Vitamin D plays a crucial role in regulation of the immune response. However, treatment of autoimmune diseases with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] doses sufficient to be effective is prohibitive due to its calcemic and toxic effects. We use the collagen-induced arthritis (CIA) model to analyze the efficacy of the noncalcemic analog of vitamin D, 20S-hydroxyvitamin D3 [20S(OH)D3], as well as 1,25(OH)2D3, to attenuate arthritis and explore a potential mechanism of action. Mice fed a diet deficient in vitamin D developed a more severe arthritis characterized by enhanced secretion of T cell inflammatory cytokines, compared to mice fed a normal diet. The T cell inflammatory cytokines were effectively suppressed, however, by culture of the cells with 20S(OH)D3. Interestingly, one of the consequences of culture with 1,25(OH)2D3 or 20S(OH)D3, was upregulation of the natural inhibitory receptor leukocyte associated immunoglobulin-like receptor-1 (LAIR-1 or CD305). Polyclonal antibodies which activate LAIR-1 were also capable of attenuating arthritis. Moreover, oral therapy with active forms of vitamin D suppressed arthritis in LAIR-1 sufficient DR1 mice, but were ineffective in LAIR-1-/- deficient mice. Taken together, these data show that the effect of vitamin D on inflammation is at least, in part, mediated by LAIR-1 and that non-calcemic 20S(OH)D3 may be a promising therapeutic agent for the treatment of autoimmune diseases such as Rheumatoid Arthritis.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-ß1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-ß1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-ß1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 µg/100 µL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 µg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

20S-Hydroxyvitamin D3, a Secosteroid Produced in Humans, Is Anti-Inflammatory and Inhibits Murine Autoimmune Arthritis.

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20S-hydroxyvitamin D3 [20S(OH)D3]. We demonstrate that 20S(OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20S(OH)D3 reduces lymphocyte subsets such as CD4+ T cells and CD19+ B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20S(OH)D3 as a potential treatment for RA and other autoimmune disorders.

Modulation by 17,20S(OH)2pD of Fibrosis-Related Mediators in Dermal Fibroblast Lines from Healthy Donors and from Patients with Systemic Sclerosis.

We previously demonstrated that the non-calcemic pregnacalciferol (pD) analog 17,20S (OH)2pD suppressed TGF-ß1-induced type I collagen production in cultured normal human dermal fibroblasts. In the present studies, we examined fibroblasts cultured from the lesional skin of patients with systemic sclerosis (scleroderma (SSc)) and assessed the effects of 17,20S(OH)2pD on fibrosis-related mediators. Dermal fibroblast lines were established from skin biopsies from patients with SSc and healthy controls. Fibroblasts were cultured with either 17,20S(OH)2pD or 1,25(OH)2D3 (positive control) with/without TGF-ß1 stimulation and extracted for protein and/or mRNA for collagen synthesis and mediators of fibrosis (MMP-1, TIMP-1, PAI-1, BMP-7, PGES, GLI1, and GLI2). 1 7,20S(OH)2pD (similar to 1,25(OH)2D3) significantly suppressed net total collagen production in TGF-ß1-stimulated normal donor fibroblast cultures and in cultures of SSc dermal fibroblasts. 17,20S(OH)2pD (similar to 1,25(OH)2D3) also increased MMP-1, BMP-7, and PGES and decreased TIMP-1 and PAI1 expression in SSc fibroblasts. Although 17,20S(OH)2pD had no effect on Gli1 or Gli2 in SSc fibroblasts, it increased Gli2 expression when cultured with TGF-ß1 in normal fibroblasts. These studies demonstrated that 17,20S(OH)2pD modulates mediators of fibrosis to favor the reduction of fibrosis and may offer new noncalcemic secosteroidal therapeutic approaches for treating SSc and fibrosis.

Retrospective Analysis of the Impact of Adalimumab Initiation on Corticosteroid Utilization and Medical Costs Among Biologic-Naïve Patients with Rheumatoid Arthritis.

INTRODUCTION: Treatment guidelines recommend low-dose corticosteroids as short-term therapy among rheumatoid arthritis (RA) patients. However, it may be difficult to wean/eliminate steroids once initiated. Initiation of more effective therapies such as biologics may help to taper corticosteroid use. The objective was to examine the impact of adalimumab (ADA) initiation on steroid utilization and non-drug medical costs among patients with RA. METHODS: A retrospective analysis was conducted among adult RA patients initiating ADA as the initial biologic in the MarketScan Database (2012-2016). Study outcomes included whether oral/injectable steroids were used, daily dose, dosage categories (< 5 and ≥ 5 mg/day), number of steroid injections, and non-drug medical costs. Outcomes were compared 6 months pre- and post-ADA initiation. Mixed effects logistic, classical linear, multinomial logistic models, and linear model with a log link and gamma distribution were used to adjust for patient demographic and health characteristics. RESULTS: The sample included 7404 ADA initiators. Compared to pre-ADA initiation, in the post-initiation period there was a reduction in proportions of patients using oral steroids (from 71.80 to 62.56%) and injectable steroids (from 34.91 to 29.88%), average daily dose of oral steroids (from 3.30 to 2.62 mg/day), patients with dose ≥ 5 mg/day (from 21.76 to 16.34%), number of injections (from 0.64 to 0.53), and non-drug medical costs (from $5356.30 to $5146.84) (P < 0.01). The multivariate analysis produced similar patterns. For example, post-ADA initiation, patients were less likely to use oral steroids [odds ratio (OR) 0.51; 95% confidence interval (CI) 0.47-0.56]; coefficient estimate for daily dose reduction was - 0.68 (95% CI - 0.81 to - 0.56); ratio estimate for medical costs was 0.91 (95% CI 0.86-0.97). CONCLUSIONS: Among patients with RA, following ADA initiation, there is a reduction in steroid utilization and dosage, and non-drug medical costs. Prospective studies should be conducted to confirm this relationship in the future.

Ameliorating effects of Gö6976, a pharmacological agent that inhibits protein kinase D, on collagen-induced arthritis.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

Sex Differences in Correlation with Gene Expression Levels between Ifi200 Family Genes and Four Sets of Immune Disease-Relevant Genes.

BACKGROUND: The HIN-200 family genes in humans have been linked to several autoimmune diseases-particularly to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recently, its human counterpart gene cluster, the Ifi200 family in mice, has been linked to spontaneous arthritis disease (SAD). However, many immune-mediated diseases (including RA and SLE) show gender difference. Understanding whether or not and how these genes play a role in sex difference in immune-mediated diseases is essential for diagnosis/treatment. METHODS: This study takes advantage of the whole genome gene expression profiles of recombinant inbred (RI) strain populations from female and male mice to analyze potential sex differences in a variety of genes in disease pathways. Expression levels and regulatory QTL of Ifi200 family genes between female and male mice were first examined in a large mouse population, including RI strains derived from C57BL/6J, DBA/2J (BXD), and classic inbred strains. Sex similarities and differences were then analyzed for correlations with gene expression levels between genes in the Ifi200 family and four selected gene sets: known immune Ifi200 pathway-related genes, lupus-relevant genes, osteoarthritis- (OA-) and RA-relevant genes, and sex hormone-related genes. RESULTS: The expression level of Ifi202b showed the most sex difference in correlation with known immune-related genes (the P value for Ifi202b is 0.0004). Ifi202b also showed gender difference in correlation with selected sex hormone genes, with a P value of 0.0243. When comparing coexpression levels between Ifi200 genes and lupus-relevant genes, Ifi203 and Ifi205 showed significant sex difference (P values: 0.0303 and 0.002, resp.). Furthermore, several key genes (e.g., Csf1r, Ifnb1, IL-20, IL-22, IL-24, Jhdm1d, Csf1r, Ifnb1, IL-20, IL-22, IL-24, and Tgfb2 that regulate sex differences in immune diseases) were discovered. CONCLUSIONS: Different genes in the Ifi200 family play different roles in sex difference among dissimilar pathways of these four gene groups.

Investigation of 20S-hydroxyvitamin D3 analogs and their 1α-OH derivatives as potent vitamin D receptor agonists with anti-inflammatory activities.

20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.

1α,20S-Dihydroxyvitamin D3 Interacts with Vitamin D Receptor: Crystal Structure and Route of Chemical Synthesis.

1α,20S-Dihydroxyvitamin D3 [1,20S(OH)2D3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1α-OH configuration. 1,20S(OH)2D3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFNγ and IL1ß). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)2D3 using the intermediate 1α,3ß-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)2D3 and its analogs as potential therapeutic agents.

Skin Exposure to Ultraviolet B Rapidly Activates Systemic Neuroendocrine and Immunosuppressive Responses.

The back skin of C57BL/6 mice was exposed to a single 400 mJ cm-2 dose of ultraviolet B (UVB), and parameters of hypothalamic-pituitary-adrenal (HPA) axis in relation to immune activity were tested after 30-90 min following irradiation. Levels of brain and/or plasma corticotropin-releasing hormone (CRH), ß-endorphin, ACTH and corticosterone (CORT) were enhanced by UVB. Hypophysectomy had no effect on UVB-induced increases of CORT. Mitogen-induced IFNγ production by splenocytes from UVB-treated mice was inhibited at 30, 90 min and after 24 h. UVB also led to inhibition of IL-10 production indicating an immunosuppressive effect on both Th1 and Th2 cytokines. Conditioned media from splenocytes isolated from UVB-treated animals had no effect on IFNγ production in cultured normal splenocytes; however, IFNγ increased with conditioned media from sham-irradiated animals. Sera from UVB-treated mice suppressed T-cell mitogen-induced IFNγ production as compared to sera from sham-treated mice. IFNγ production was inhibited in splenocytes isolated from UVB-treated animals with intact pituitary, while stimulated in splenocytes from UVB-treated hypophysectomized mice. Thus, cutaneous exposure to UVB rapidly stimulates systemic CRH, ACTH, ß-endorphin and CORT production accompanied by rapid immunosuppressive effects in splenocytes that appear to be independent of the HPA axis.

Design, Synthesis and Biological Activities of Novel Gemini 20S-Hydroxyvitamin D3 Analogs.

Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.

Chemical Synthesis and Biological Activities of 20S,24S/R-Dihydroxyvitamin D3 Epimers and Their 1α-Hydroxyl Derivatives.

Bioactive vitamin D3 metabolites 20S,24S-dihydroxyvitamin D3 [20S,24S(OH)2D3] and 20S,24R-dihydroxyvitamin D3 [20S,24R(OH)2D3] were chemically synthesized and confirmed to be identical to their enzymatically generated counterparts. The absolute configurations at C24 and its influence on the kinetics of 1α-hydroxylation by CYP27B1 were determined. Their corresponding 1α-hydroxyl derivatives were subsequently produced. Biological comparisons of these products showed different properties with respect to vitamin D3 receptor activation, anti-inflammatory activity, and antiproliferative activity, with 1α,20S,24R(OH)2D3 being the most potent compound.

Differences between Mice and Humans in Regulation and the Molecular Network of Collagen, Type III, Alpha-1 at the Gene Expression Level: Obstacles that Translational Research Must Overcome.

Collagen, type III, alpha-1 (COL3A1) is essential for normal collagen I fibrillogenesis in many organs. There are differences in phenotypes of mutations in the COL3A1 gene in humans and mutations in mice. In order to investigate whether the regulation and gene network of COL3A1 is the same in healthy populations of mice and humans, we compared the quantitative trait loci (QTL) that regulate the expression level of COL3A1 and the gene network of COL3A1 pathways between humans and mice using whole genome expression profiles. Our results showed that, for the regulation of expression of Col3a1 in mice, an eQTL on chromosome (Chr) 12 regulates the expression of Col3a1. However, expression of genes in the syntenic region on human Chr 7 has no association with the expression level of COL3A1. For the gene network comparison, we identified 44 top genes whose expression levels are strongly associated with that of Col3a1 in mice. We next identified 41 genes strongly associated with the expression level of COL3A1 in humans. There are a few but significant differences in the COL3A1 gene network between humans and mice. Several genes showed opposite association with expression of COL3A1. These genes are known to play important roles in development and function of the extracellular matrix of the lung. Difference in the molecular pathway of key genes in the COL3A1 gene network in humans and mice suggest caution should be used in extrapolating results from models of human lung diseases in mice to clinical lung diseases in humans. These differences may influence the efficacy of drugs in humans whose development employed mouse models.

Pathogenesis of Systemic Sclerosis.

Systemic scleroderma (SSc) is one of the most complex systemic autoimmune diseases. It targets the vasculature, connective tissue-producing cells (namely fibroblasts/myofibroblasts), and components of the innate and adaptive immune systems. Clinical and pathologic manifestations of SSc are the result of: (1) innate/adaptive immune system abnormalities leading to production of autoantibodies and cell-mediated autoimmunity, (2) microvascular endothelial cell/small vessel fibroproliferative vasculopathy, and (3) fibroblast dysfunction generating excessive accumulation of collagen and other matrix components in skin and internal organs. All three of these processes interact and affect each other. The disease is heterogeneous in its clinical presentation that likely reflects different genetic or triggering factor (i.e., infection or environmental toxin) influences on the immune system, vasculature, and connective tissue cells. The roles played by other ubiquitous molecular entities (such as lysophospholipids, endocannabinoids, and their diverse receptors and vitamin D) in influencing the immune system, vasculature, and connective tissue cells are just beginning to be realized and studied and may provide insights into new therapeutic approaches to treat SSc.

Comparison of LAIR-1 genetic pathways in murine vs human internal organs.

Growing evidence suggests that defective expression or dysfunction of LAIR-1, a novel immunoinhibitory receptor for collagen, is closely associated with some autoimmune diseases, cancers, as well as viral infections. We analyzed the variation of LAIR-1 genetic pathways in murine versus human internal organs, including the lung and brain. The results showed that, under physiological conditions, LAIR-1 links more closely to the common genes in mouse than in human, which poses tissue specificity. It means that mice experimental data in relation to the role of LAIR-1 immune regulation may be overestimated when applied to assess human conditions. Moreover, we found that the in vivo interaction of LAIR-1 with LAIR-2 rarely occurs, implying that the species difference in LAIR-1 genetic pathways could not be primarily attributed to the existence of human LAIR-2. In summary, this study opens the door for insight into LAIR-1 functions inside the human body, and raises concern as to extrapolative credibility of the murine model in biomedical research.

Chance, genetics, and the heterogeneity of disease and pathogenesis in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a remarkably complex and heterogeneous systemic autoimmune disease. Disease complexity within individuals and heterogeneity among individuals, even genetically identical individuals, is driven by stochastic execution of a complex inherited program. Genome-wide association studies (GWAS) have progressively improved understanding of which genes are most critical to the potential for SLE and provided illuminating insight about the immune mechanisms that are engaged in SLE. What initiates expression of the genetic program to cause SLE within an individual and how that program is initiated remains poorly understood. If we extrapolate from all of the different experimental mouse models for SLE, we can begin to appreciate why SLE is so heterogeneous and consequently why prediction of disease outcome is so difficult. In this review, we critically evaluate extrinsic versus intrinsic cellular functions in the clearance and elimination of cellular debris and how dysfunction in that system may promote autoimmunity to nuclear antigens. We also examine several mouse models genetically prone to SLE either because of natural inheritance or inheritance of induced mutations to illustrate how different immune mechanisms may initiate autoimmunity and affect disease pathogenesis. Finally, we describe the heterogeneity of disease manifestations in SLE and discuss the mechanisms of disease pathogenesis with emphasis on glomerulonephritis. Particular attention is given to discussion of how anti-DNA autoantibody initiates experimental lupus nephritis (LN) in mice.

Characterization of T cell phenotype and function in a double transgenic (collagen-specific TCR/HLA-DR1) humanized model of arthritis.

INTRODUCTION: T cells orchestrate joint inflammation in rheumatoid arthritis (RA), yet they are difficult to study due to the small numbers of antigen-specific cells. The goal of this study was to characterize a new humanized model of autoimmune arthritis and to describe the phenotypic and functional changes that occur in autoimmune T cells following the induction of pathological events. METHODS: We developed a double transgenic mouse containing both the HLA-DR1 transgene and an HLA-DR1-restricted collagen-specific TCR in order to obtain large numbers of antigen-specific T cells that can be used for immunologic studies. RESULTS: In vitro, CII-specific T cells from this mouse proliferated vigorously in response to the CII immunodominant peptide A2 and the cells altered their phenotype to become predominately CD62Llow and CD44high "activated" T cells. The response was accompanied by the production of Th1, Th2, and Th17-type cytokines. Following immunization with bovine CII/CFA, these mice develop an accelerated arthritis compared to single transgenic HLA-DR1 mice. On the other hand, when the mice were treated orally with the analog peptide A12, (a suppressive analog of collagen we have previously described), arthritis was significantly suppressed, despite the fact that >90% of the CD4+ T cells express the TCR Tg. In GALT tissues taken from the A12-treated mice, IL-2, IFN-γ, and IL-17 production to the autoimmune collagen determinant dropped while high levels of IL-10 and IL-4 were produced. CONCLUSIONS: We have developed a humanized model of autoimmune arthritis that will be useful for the study of T cell directed therapies as well as T cell mediated mechanisms of autoimmune diseases.

20S-hydroxyvitamin D3, noncalcemic product of CYP11A1 action on vitamin D3, exhibits potent antifibrogenic activity in vivo.

CONTEXT: There is no effective treatment for systemic sclerosis and related fibrosing diseases. Recently the action of CYP11A1 on vitamin D(3) was shown to produce biologically active 20S-hydroxyvitamin D [20(OH)D(3)] and 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), and 17,20,23(OH)(3)D(3). OBJECTIVES: Because 20(OH)D(3) is noncalcemic (nontoxic) in vivo at very high doses, we evaluated its antifibrogenic activities both in vitro and in vivo. Because it is further metabolized by CYP11A1, we also tested preclinical utilities of its hydroxyderivatives, especially 20,23(OH)(2)D(3). DESIGN: Human dermal fibroblasts from scleroderma and normal donors were used to test the efficiency of hydroxyvitamin D derivatives in inhibiting TGF-ß1-induced collagen and hyaluronan synthesis and inhibiting cell proliferation. The in vivo activity of 20(OH)D(3) was tested using bleomycin-induced sclerosis in C57BL/6 mice. RESULTS: 20(OH)D(3) and 20,23(OH)(2)D(3) inhibited TGF-ß1-induced collagen and hyaluronan synthesis similarly to 1,25(OH)(2)D(3) in cultured human fibroblasts. Also, 20(OH)D(3), 20,23(OH)(2)D(3), and 1,25(OH)(2)D(3) suppressed TGF-ß1-induced expression of COL1A2, COL3A1, and hyaluronan synthase-2 mRNA, indicating that they regulate these matrix components at the transcriptional level. 20(OH)D(3), 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), and 17,20,23(OH)(3)D(3) inhibited proliferation of dermal fibroblasts with comparable potency with 1,25(OH)(2)D(3), with 20(OH)D(2) being less active and 1α(OH)D(3) being almost inactive. 20,23(OH)(2)D(3) at 3 µg/kg had no effect on serum Ca(++) or fibroblast growth factor-23 levels and did not cause any noticeable signs of morbidity. 20(OH)D(3) markedly suppressed fibrogenesis in mice given sc bleomycin as demonstrated by total collagen content and hematoxylin and eosin staining of skin biopsies. CONCLUSIONS: 20(OH)D(3) is an excellent candidate for preclinical studies on scleroderma, with other CYP11A1-derived products of its metabolism deserving further testing for antibrogenic activity.

20-hydroxyvitamin D3 inhibits proliferation of cancer cells with high efficacy while being non-toxic.

AIM: To define the potential utility of 20-hydroxyvitamin D(3) (20(OH)D(3)) as a tumorostatic agent, we assessed its in vitro antiproliferative activity and its in vivo toxicity. MATERIALS AND METHODS: The antitumor activity of 20(OH)D(3) was tested against breast and liver cancer cell lines using colony formation assays. To assess in vivo toxicity, mice were injected with 5-30 µg/kg 20(OH)D(3) intraperitoneally each day for 3 weeks. Blood and organ samples were collected for clinical pathology analyses. RESULTS: 20(OH)D(3) displays similar tumorostatic activity towards MDA-MB-453 and MCF7 breast carcinomas, and HepG2 hepatocarcinoma, in a dose-dependent manner. This compound is not hypercalcemic, does not cause detectable toxicities in liver, kidney, or blood chemistry in mice at a dose as high as 30 µg/kg. In contrast, both 25(OH)D(3) and 1,25(OH)(2)D(3) caused severe hypercalcemia at a dose of 2 µg/kg. CONCLUSION: 20(OH)D(3) possesses high efficacy for inhibiting cancer cell proliferation in vitro and is non-toxic in vivo, supporting its further development as a potential anticancer therapeutic agent.