VTP-195183

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.

All-trans retinoic acid (ATRA) is used successfully in the treatment of acute promyelocytic leukemia (APL). ATRA enhances hematopoietic stem cell self-renewal through retinoic acid receptor (RAR)γ activation while promoting differentiation of committed myeloid progenitors through RARα activation. Its lack of success in the treatment of non-APL acute myeloid leukemia (AML) may be related to ATRA's non-selectivity for the RARα and RARγ isotypes, and specific RARα activation may be more beneficial in promoting myeloid differentiation. To investigate this hypothesis, the effects of ATRA and the specific RARα agonist NRX195183 was assessed in AML1-ETO (AE)-expressing murine bone marrow (BM) progenitors. ATRA potentiated the in vitro clonogenicity of these cells while NRX195183 had the opposite effect. Morphological and flow cytometric analysis confirmed a predominantly immature myeloid population in the ATRA-treated AE cells while the NRX195183-treated cells demonstrated an increase in the mature myeloid population. Similarly, NRX195183 treatment promoted myeloid differentiation in an AE9a in vivo murine model. In the ATRA-treated AE cells, gene expression analyses revealed functional networks involving SERPINE1 and bone morphogenetic protein 2; AKT phosphorylation was upregulated. Collectively, these findings confirm the contrasting roles of specific RARα and RARγ activation in the clonogenicity and differentiation of AE cells with potential significant implications in the treatment of non-APL AML using a specific RARα agonist.