The effects of all-trans retinoic acid on prednisolone-induced osteoporosis in zebrafish larvae

Abstract

Glucocorticoids (GCs) are extensively used as anti-inflammatory and immunosuppressive medications in the long-term treatment of rheumatic disorders, respiratory diseases, renal diseases, and organ transplantation. Prolonged use of GCs can reduce bone mineral density, leading to osteoporosis (Glucocorticoid Induced Osteoporosis, GIOP) and fracture. All-trans retinoic acid (ATRA) is an active vitamin A metabolite that regulates embryonic development and adult organ function. ATRA has been found in studies to enhance osteogenesis. To examine the interventional effects of ATRA on GIOP and the mechanisms of ATRA activities, we first performed bioinformatic analysis to identify potential gene targets of ATRA. Zebrafish larvae were recruited as experimental animals, and the frequently used GC, prednisolone, was administered to larvae to construct a GIOP model. We evaluated the influence of exogenous ATRA on the activities of bone metabolic enzymes, the expression of genes linked to osteoblasts and osteoclasts, and the restoration of bone mineral density and bone mass in GIOP zebrafish larvae. Furthermore, we studied the influence of RBM14, a transcriptional coactivator and negative reciprocal factor of ATRA, on the regulation of osteoblastic gene expression during the anti-GIOP process of ATRA using the morpholino knockdown approach. The findings of bone metabolic enzyme activity (alkaline phosphatase, ALP and tartrate-resistant acid phosphatase, TRAP) and expression assays of osteoblastic marker genes (Runx2a, Runx2b, SP7, Csf1a, RANKL, and CTSK) indicated that ATRA had bidirectional effects on osteogenesis. However, in the GIOP model, ATRA reversed the GIOP-induced osteoporosis phenotype by inhibiting the GIOP-induced suppression of osteoblastic metabolic enzyme (ALP) activities and osteoblastic marker gene expression (Runx2a, Runx2b, and SP7), and this antagonism was concentration-dependent. We also observed that ATRA inhibited RBM14 expression in zebrafish larvae, while ATRA alone and RBM14 knockdown showed a consistent induction of osteoblast marker gene expression, implying that ATRA's inhibitory effect on RBM14 expression may underlie ATRA's osteogenic effects. Based on these data, we postulated that ATRA may ameliorate GIOP by decreasing RBM14 expression, thereby enhancing osteoblastic marker gene expression.