Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH)₂D₃, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.     

Retinoic acid via RARalpha inhibits the expression of 24-hydroxylase in human prostate stromal cells

25-Hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) is an important inactivating enzyme and its expression is induced by 25-hydroxyvitamin D3 (25OHD3) and 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) through action of heterodimers of vitamin D receptor (VDR) and retinoid X receptor (RXR). RXRs also act as heterodimer partners for retinoic acid receptors (RARs), mediating the action of all-trans-retinoic acid (ATRA). Prostate stroma plays a crucial role in prostate cancer development and benign prostatic hyperplasia. We demonstrate here that ATRA markedly reduced the expression of 24-hydroxylase mRNA induced by 25OHD3 and 1alpha,25-(OH)2D3 in human prostatic stromal cells P29SN and P32S but not in epithelial cells PrEC or cancer cells LNCaP. By using transfection and RAR-selective ligands, we found that the inhibitory effect of ATRA on 24-hydroxylase expression in stromal cells was mediated by RARalpha but not by RARbeta. Moreover, the ATRA-induced expression of RARbeta was also mediated by RARalpha. The combined treatment of 1alpha,25-(OH)2D3 and RARalpha agonist Am80 at 10 nM exhibited strong growth-inhibitory effect whereas either alone had no effect. Our data suggest that ATRA suppresses 24-hydroxylase expression through RARalpha-dependent signaling pathway and can enhance vitamin D action in suppression of cell growth.     

Multiplicity generates diversity in the retinoic acid signalling pathways.

Complexity in the retinoid signalling system arises from a combination of several forms of retinoic acid, multiple cytoplasmic binding proteins and nuclear receptors, and the existence of polymorphic retinoic acid response elements. Additional diversity appears to be generated by heterodimeric interactions between two families of nuclear retinoic acid receptors, and between nuclear retinoic acid receptors and other members of the nuclear receptor superfamily. Thus, a complex array of combinatorial effects is beginning to emerge which may account for the pleiotropic effects of retinoids.     

The flavonoid apigenin suppresses vitamin D receptor expression and vitamin D responsiveness in normal human keratinocytes

Apigenin, a flavonoid with chemopreventive properties, induces cellular growth arrest, with concomitant inhibition of intracellular signaling cascades and decreased proto-oncogene expression. We report that apigenin potently inhibited vitamin D receptor (VDR) mRNA and protein expression in human keratinocytes without changes in VDR mRNA half-life. Concurrently, downregulation of retinoid X receptor alpha, a dramatic loss of c-myc mRNA, and upregulation of p21(WAF1) took place. Furthermore, a nearly complete suppression of vitamin D responsiveness was observed as estimated by induction of 24-hydroxylase mRNA. The apigenin effect on VDR expression was shared by some other (quercetine and fisetine) but not all tested flavonoids. Interestingly, the apigenin-mediated VDR suppression was counteracted by the NFkappaB inhibitors sodium salicylate and caffeic acid phenethyl ester. The presented results propose suppression of nuclear receptor levels as a novel mechanism whereby flavonoids exert their pleiotropic effects. This study may also contribute to the understanding of the regulation of VDR expression in epidermal keratinocytes.     

Metabolism and transactivation activity of 13,14-dihydroretinoic acid

The metabolism of vitamin A is a highly regulated process that generates essential mediators involved in the development, cellular differentiation, immunity, and vision of vertebrates. Retinol saturase converts all-trans-retinol to all-trans-13,14-dihydroretinol (Moise, A. R., Kuksa, V., Imanishi, Y., and Palczewski, K. (2004) J. Biol. Chem. 279, 50230-50242). Here we demonstrate that the enzymes involved in oxidation of retinol to retinoic acid and then to oxidized retinoic acid metabolites are also involved in the synthesis and oxidation of all-trans-13,14-dihydroretinoic acid. All-trans-13,14-dihydroretinoic acid can activate retinoic acid receptor/retinoid X receptor heterodimers but not retinoid X receptor homodimers in reporter cell assays. All-trans-13,14-dihydroretinoic acid was detected in vivo in Lrat-/- mice supplemented with retinyl palmitate. Thus, all-trans-13,14-dihydroretinoic acid is a naturally occurring retinoid and a potential ligand for nuclear receptors. This new metabolite can also be an intermediate in a retinol degradation pathway or it can serve as a precursor for the synthesis of bioactive 13,14-dihydroretinoid metabolites.     

Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.