VTP-195183

Activities of the retinoic acid receptor (RAR)α and RARγ are important to hematopoiesis. Here, we have investigated the effects of receptor selective agonists and antagonists on the primitive human hematopoietic cell lines KG1 and NB-4 and purified normal human hematopoietic stem cells (HSCs). Agonizing RARα (by AGN195183) was effective in driving neutrophil differentiation of NB-4 cells and this agonist synergized with a low amount (10 nM) of 1α,25-dihydroxyvitamin D₃ to drive monocyte differentiation of NB-4 and KG1 cells. Treatment of cultures of human HSCs (supplemented with stem cell factor ± interleukin 3) with an antagonist of all RARs (AGN194310) or of RARα (AGN196996) prolonged the lifespan of cultures, up to 55 days, and increased the production of neutrophils and monocytes. Slowing down of cell differentiation was not observed, and instead, hematopoietic stem and progenitor cells had expanded in number. Antagonism of RARγ (by AGN205728) did not affect cultures of HSCs. Studies of CV-1 and LNCaP cells transfected with RAR expression vectors and a reporter vector revealed that RARγ and RARβ are activated by sub-nM all-trans retinoic acid (EC₅₀-0.3 nM): ~50-fold more is required for activation of RARα (EC₅₀-16 nM). These findings further support the notion that the balance of expression and activity of RARα and RARγ are important to hematopoietic stem and progenitor cell expansion and differentiation.

Retinoids, including all‐trans retinoic acid (tRA), have dose‐dependent pro‐fibrotic effects in experimental kidney diseases. To understand and eventually prevent such adverse effects, it is important to establish relevant in vitro models and unravel their mechanisms.

Fibrogenic effects of retinoids were assessed in NRK‐49F renal fibroblasts using picro‐Sirius red staining for collagens and quantified by spectrophotometric analysis of the eluted stain. Other methods included RT‐qPCR, immunoassays and matrix metalloproteinase (MMP) activity assays.

With or without TGF‐β1, tRA was dose‐dependently pro‐fibrotic, notably increasing collagen accumulation. tRA and TGF‐β1 additively suppressed expression of mRNA for MMP2, 3 and 13 and suppressed MMP activity. tRA, in the presence of TGF‐β1, induced plasminogen activator inhibitor‐1 (PAI‐1) mRNA and they additively induced PAI‐1 protein expression. A PAI‐1 inhibitor, a pan‐retinoic acid receptor (RAR) antagonist and a pan‐retinoid X receptor (RXR) antagonist each partially prevented the pro‐fibrotic effect of tRA. The dose‐dependent pro‐fibrotic effects of a pan‐RXR agonist were similar to those of tRA. A pan‐RAR agonist showed weaker, less dose‐dependent pro‐fibrotic effects and the pro‐fibrotic effects of RARα and RARβ‐selective agonists were even smaller. An RARγ‐selective agonist did not affect fibrogenesis.

An in vitro model for the pro‐fibrotic effects of retinoids was established in NRK‐49F cells. It was associated with reduced MMP activity and increased PAI‐1 expression, and was probably mediated by RXR and RAR. To avoid or antagonize the pro‐fibrotic activity of tRA, further studies on RAR isotype‐selective agonists and PAI‐1 inhibitors might be of value.