Reversal of myofibroblast differentiation by phorbol 12‐myristate 13‐acetate is PKC‐independent

Abstract

Fibrosis often results in organ dysfunction and failure in diseases, such as: chronic heart failure, hepatic cirrhosis, pulmonary fibrosis, and end‐stage renal disease. The differentiation of fibroblasts into myofibroblasts results in the secretion of collagens and other extracellular matrix proteins that limit organ function and underlie the fundamental basis of fibrosis. TGF‐β1 induces the differentiation of α‐SMA‐expressing myofibroblasts from fibroblasts. However, the mechanism to induce reversal of myofibroblast differentiation remains elusive. Phorbol 12‐myristate 13‐acetate (PMA) has been used for the stimulation of lymphocytes and splenocytes. It is also involved in multiple cellular functions by potently activating protein kinase C (PKC). Nevertheless, the effect of PMA on the formation of myofibroblasts is unknown. To investigate whether PMA plays a role in myofibroblast differentiation, NIH 3T3 fibroblasts were cultured in DMEM medium for 48 hours in the presence of DMSO (vehicle control), PMA, or PKC inhibitors (calphostin C, chelerythrine, and staurosporine) alone or plus PMA. The expression of α‐smooth muscle actin (SMA, a hallmark of the myofibroblastic phenotype), fibroblast‐specific protein 1 (FSP1, a biomarker of fibroblasts), and TGF‐β1 in cultured fibroblasts was detected by western blotting and immunofluorescence. Expression of α‐MARCK phosphorylation was also detected to examine the activity of PKC activation. Positive staining of α‐SMA was observed in some cells in control culture conditions, and these α‐SMA positive cells exhibited significant morphology changes with large nuclei and cytoplasm, compared with α‐SMA negative fibroblasts. Treatment with PMA 50 ng/mL for 48 hours reduced the expression of α‐SMA and TGF‐β1, and pretreatment with the three PKC inhibitors mentioned above could not abrogate PMA‐induced reduction of α‐SMA and TGF‐β1. These results indicate that some myofibroblasts are derived from fibrolbasts under basic culture conditions. PMA induces reversal of myofibroblast differentiation via a PKC‐independent mechanism.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.