Paul N. MacDonald

Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene.

The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Retinoic acid suppresses parathyroid hormone (PTH) secretion and PreproPTH mRNA levels in bovine parathyroid cell culture.

1,25-dihydroxyvitamin D3[1,25(OH)2D3] suppresses parathyroid hormone (PTH) gene transcription. Recent evidence suggests that retinoid X receptors are involved in 1,25(OH)2D3-mediated transcriptional events. However, little data exists for a role of retinoids in parathyroid function or in PTH expression. In the present study, we observed that all-trans- or 9-cis retinoic acid suppressed the release of PTH from bovine parathyroid cell cultures. Both retinoids were remarkably potent with significant decreases evident at 10(-10) M and a maximally suppressive effect (approximately 65%) at 10(-7) M. All-trans-retinol was considerably less potent in this system. The effect was not evident until 12 h, suggesting that retinoids did not affect the rapid secretion of preexisting PTH stores. PreproPTH mRNA levels were also suppressed by retinoic acid and the retinoid potencies were similar to those observed in the secretion studies. Combined treatment with 10(-6) M retinoic acid and 10(-8) M 1,25(OH)2D3 more effectively decreased PTH secretion and preproPTH mRNA than did either compound alone. These data indicate that retinoic acid: (a) elicits a bioresponse in bovine parathyroid cells; (b) attenuates PTH expression at the protein and mRNA levels, and (c) acts independently of 1,25(OH)2D3 in the control of PTH expression.

Assay of lecithin-retinol acyltransferase.

Publisher Summary Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II (CRBP II) are two of several binding proteins that carry retinol as an endogeneous ligand in vivo . Retinol complexed to CRBP and CRBP II are used as substrates for the in vitro esterifications described in this chapter. Both complexes are effective substrates for lecithin-retinol acyltransferase (LRAT). This chapter described the methods used to examine retinol esterification by LRAT. Two in vitro assays are routinely used by this laboratory to characterize LRAT. The procedures differ mainly in the method of analysis. When the individual retinyl esters synthesized in the assay are of interest, the products are analyzed by an HPLC system that separates the retinyl esters based on their acyl chain length and degree of saturation. If the total amount of retinyl ester synthesis is the only concern, then [ 3 H]retinol-CRBP II or [ 3 H]retinol-CRBP are used as substrates and the total [ 3 H]retinyl esters synthesized are determined after batch elution from columns of aluminum oxide.