CARD-024, a vitamin D analog, attenuates the pro-fibrotic response to substrate stiffness in colonic myofibroblasts

Abstract

Intestinal fibrosis is one of the major complications of Crohn's disease (CD) for which there are no effective pharmacological therapies. Vitamin D deficiency is common in CD, though it is not known whether this is a contributing factor to fibrosis, or simply a consequence of the disease itself. In CD, fibrosis is mediated mainly by activated intestinal myofibroblasts during remodeling of extracellular matrix in response to wound healing. We investigated the effects of CARD-024 (1-alpha-hydroxyvitamin D5), a vitamin D analog with minimal hypercalcemic effects, on the pro-fibrotic response of intestinal myofibroblasts to two fibrogenic stimuli: TGFβ stimulation and culture on a physiologically stiff matrix. TGFβ stimulated a fibrogenic phenotype in Ccd-18co colonic myofibroblasts, characterized by an increase in actin stress fibers and mature focal adhesions, and increased αSMA protein expression, while CARD-024 repressed αSMA protein expression in a dose-dependent manner. Culture of colonic myofibroblasts on physiological high stiffness substrates induced morphological changes with increased actin stress fibers and focal adhesion staining, induction of αSMA protein expression, FAK phosphorylation, induction of fibrogenic genes, and repression of COX-2 and IL-1β. CARD-024 treatment repressed the stiffness-induced morphological features including stellate cell morphology and the maturation of focal adhesions. CARD-024 repressed the stiffness-mediated induction of αSMA protein expression, FAK phosphorylation, and MLCK and ET-1 gene expression. In addition, CARD-024 partially stimulated members of the COX-2/IL-1β inflammatory pathway. In summary, CARD-024 attenuated the pro-fibrotic response of colonic myofibroblasts to high matrix stiffness, suggesting that vitamin D analogs such as CARD-024 may ameliorate intestinal fibrosis.