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Vitamin D plays a pivotal role to maintain skeletal muscle integrity and health. Vitamin D deficiency characterizes inflammatory myopathy (IM) and diabetes, often overlapping diseases involving skeletal muscle damage. Vitamin D receptor (VDR) agonists likely exert beneficial effects in both IM and metabolic disturbances. We aim to evaluate in vitro the effect of elocalcitol, a non-hypercalcemic VDR agonist, on the biomolecular metabolic machinery of human skeletal muscle cells (Hfsmc), vs. insulin (I). We analyzed GLUT4, Flotillin-1, Caveolin-3 and Caveolin-1 cell expression/localization; mTOR, AKT, ERK and 4E-BP1 phosphorylation; IL-6 myokine release; VDR expression. We investigated in vivo vitamin D status in IM subjects, evaluating VDR muscular expression and serum vitamin D with metabolism-related parameters, as glycemia, triglycerides, cholesterol, resistin and adiponectin. In Hfsmc, elocalcitol exerted an I-like effect, promoting GLUT4 re-localization in Flotillin-1, Caveolin-3 and Caveolin-1 positive sites and mTOR, AKT, ERK, 4E-BP1 activation; it enhanced IL-6 myokine release. IM subjects, all normoglycemic, showed VDR/vitamin D deficiency that, together with high lipidemic and resistin profile, possibly increases the risk to develop metabolic diseases. VDR agonists as elocalcitol may be therapeutic tools for skeletal muscle integrity/function maintenance, an indispensable condition for health homeostasis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The primary aim in the treatment of autoimmune inflammatory myopathies (IMs) is to recover muscle function. The presence of immune/inflammatory cell infiltrates within muscle tissues represents the common feature of different IM subtypes, albeit a correlation between muscular damage extent and inflammation degree is often lacking. Treatments for IMs are based on life-long immunosuppressive therapy, with the well known adverse effects; recovery is incomplete for many patients. More effective therapies, with reduced side-effects, are highly desirable. Vitamin D receptor (VDR) agonists emerge to retain pleiotropic anti-inflammatory properties, since they regulate innate and adaptive immunity by switching the immune response from proinflammatory T helper 1 (Th1) type to tolerogenic T helper 2 (Th2) type dominance. In skeletal muscle cells less hypercalcemic VDR ligands target powerful mediators of inflammation, such as TNF α and TNF α driven paths, without affecting immune or muscle cells viability, retaining the potentiality to counteract Th1 driven overreactivity established by the self-enhancing inflammatory loop between immune and skeletal muscle cells. This review summarizes those features of VDR agonists as candidates in future treatment of IM.

Objective: This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects., Methods: Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC50 determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis., Results: BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls., Conclusions: Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.

Understanding the many biological extraskeletal actions of vitamin D has increased in the past decades. Indeed, vitamin D and analogue molecules, besides the classical actions on bone metabolism, exert several beneficial effects on metabolic homeostasis, heart-cardiovascular, brain, and muscle physiological functions, throughout the interaction with the specific vitamin D receptor (VDR). In particular, VDR agonists powerfully control innate and adaptive immune system with favorable effects on human health. VDR ligands act as immunomodulators that are potent enough to retain anti-inflammatory effects, even though the mechanism underlying those effects is not yet fully elucidated. VDR agonists exert a significant suppression of inflammatory processes switching the immune response from T helper 1 (Th1) to T helper 2 (Th2) dominance and counteracting the self-enhancing inflammatory loop between immune and resident cells, especially by cytokine release impairment. Those molecules are able, indeed, to reduce the release of the interferon (IFN)γ-induced 10 kDa protein IP-10/CXCL10, a powerful chemokine driving Th1-mediated inflammation. Based on their features, VDR ligands show the potentiality to be included in immunosuppressive regimens, aimed to control auto- and alloimmune Th1-driven overreactivity, occurring, for example, in autoimmune disease or graft rejection.

During kidney allograft rejection, CXC chemokine ligand 10 (CXCL10)-CXC chemokine receptor 3 (CXCR3) trafficking between peripheral blood and tissues initiates alloresponse and perpetuates a self-inflammatory loop; thus, CXCL10-CXCR3 axis could represent a pharmacologic target. In this perspective, immunosuppressors targeting graft-resident cells, beside immune cells, could be very advantageous. Vitamin D receptor (VDR) agonists exhibit considerable immunomodulatory properties. This study aimed to investigate whether elocalcitol and BXL-01-0029 could decrease the expression of CXCL10 in activated renal tubular cells in vitro and thus be useful in kidney allograft rejection treatment. Experiments were performed in human tubular renal cells stimulated with interferon-gamma + tumor necrosis factor-alpha with and without VDR agonists, tacrolimus, sirolimus, hydrocortisone, methylprednisolone, cyclosporin A and mycophenolate mofetil. CXCL10 protein secretion and gene expression were measured by ELISA and by quantitative PCR. Specific inhibitors were used to investigate intracellular pathways involved in tubular cells activation. For IC(50) determination and comparison, dose-response curves with VDR agonists, tacrolimus and mycophenolic acid were performed. Elocalcitol and BXL-01-0029 inhibited CXCL10 secretion by renal cells, without affecting cell viability, while almost all the immunosuppressors were found to be ineffective, except for tacrolimus and mycophenolate mofetil. BXL-01-0029 was the most potent drug and, notably, it was found to be capable of allowing reduction in tacrolimus-inhibitory doses. Our data suggest that BXL-01-0029 could potentially be a dose-reducing agent for conventional immunosuppressors in kidney rejection management.

Background: BXL-628 is a calcitriol analog shown to decrease prostate growth in preclinical and clinical studies. BPH symptoms are generated not only by prostate overgrowth but also by bladder overactivity, resulting from an increased RhoA/Rho-kinase signaling. Because bladder smooth muscle cells express VDR, we studied effects of BXL-628 on this pathway., Methods: RhoA and Rho-kinase gene expression and functional activity were studied in rat and human bladder smooth muscle by real-time RT-PCR, immuno-kinase assays, western blot analysis, confocal microscopy, in vitro contractility, and cell migration., Results: In bladder smooth muscle, carbachol responsiveness was delayed and Rho-kinase activity reduced by BXL-628 treatment because of impaired RhoA membrane translocation and activation. Accordingly, RhoA-mediated biological functions, such as cell migration and cytoskeleton remodeling were also inhibited by BXL-628., Conclusions: BXL-628 inhibits RhoA/Rho-kinase signaling, a calcium sensitizing pathway, suggesting its possible clinical use in the treatment of altered bladder contractility often associated with BPH-induced lower urinary tract symptoms., ((c) 2006 Wiley-Liss, Inc.)

The active form of vitamin D, 1,25-dihydroxyvitamin D3, is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the superfamily of nuclear receptors, and exerts a number of diverse biological functions. The natural hormone and synthetic VDR agonists are well known for their capacity to control calcium and bone metabolism, but they also regulate proliferation and differentiation of many cell types, and possess exquisite immunoregulatory properties, mostly by targeting dendritic cells (DC) and T cells. These properties have been clinically exploited in the treatment of different diseases, from secondary hyperparathyroidism to osteoporosis to psoriasis. The VDR is expressed by most cell types, including cells of the urogenital system such as prostate and bladder cells. In particular, the prostate has been recognized as a target organ of VDR agonists and represents an extra-renal synthesis site of 1,25-dihydroxyvitamin D3, but its capacity to respond to VDR agonists has, so far, been probed only for the treatment of prostate cancer. We have taken a different approach, and have analysed the capacity of VDR agonists to treat benign prostatic hyperplasia (BPH), a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary irritative symptoms, and a possible inflammatory component. Pre-clinical data reviewed here demonstrate that VDR agonists, and notably BXL-628 (Elocalcitol), reduce the static component of BPH by inhibiting the activity of intra-prostatic growth factors downstream of the androgen receptor, and the dynamic component by targeting bladder cells. These data have led to a proof-of-concept clinical study that has successfully shown arrest of prostate growth in BPH patients treated with BXL-628. Ongoing clinical studies will assess the capacity of this VDR agonist to reduce symptoms and ameliorate flow parameters in BPH-affected individuals. The pronounced effects of BXL-628 on bladder smooth muscle cells and its anti-inflammatory properties indeed anticipate beneficial effects also on BPH-related lower urinary tract symptoms.

Human prostate is now considered a target for vitamin D receptor (VDR) ligands, such as BXL-628. Because BXL-628 inhibited prostate growth without interfering with androgen signaling, it represents a new option for benign prostate hyperplasia (BPH) therapy. However, BPH symptoms are related not only to prostate size, but also to compensatory bladder hypertrophy and eventual overactivity. We now report that human bladder expresses VDR (determined by real-time PCR immunohistochemistry and Western blot) and responds to VDR agonists, such as the natural ligand, calcitriol, and its synthetic and less hypercalcemic derivative, BXL-628. Experiments were conducted with stromal cells derived from human bladder neck obtained at surgery from BPH patients. BXL-628 counteracted keratinocyte growth factor (KGF) and androgen-induced cell proliferation and stimulated apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-2. Prolonged serum starvation time-dependently pushed bladder stromal cells to express activated myofibroblast markers, such as desmin and smoothelin, without changing other contractile-related proteins and intermediate filaments, such as vimentin. Chronic exposure to BXL-628 prevented starvation-induced cell phenotype modification. Because hypertrophy and starvation-induced bladder remodeling are supposed to underlie bladder overactivity, it is possible that BXL-628 might be helpful in reducing not only cumbersome symptoms related to prostate overgrowth, but also those related to bladder irritation.