Retinoic acid upregulates cone arrestin expression in retinoblastoma cells through a Cis element in the distal promoter region

Invest Ophthalmol Vis Sci. 2002 May;43(5):1375-83.

Abstract

Purpose: This study was initiated to investigate the molecular mechanisms of activation of expression of the human cone arrestin (hCAR) gene by retinoic acid (RA), in an in vitro model of retinoblastoma cells.

Methods: Human retinoblastoma Weri-Rb-1 or Y79 cell lines were cultured in the absence or presence of RA analogues with transcription or translation inhibitors for various periods. The mRNAs encoding hCAR, retinoic acid receptor (RAR), and retinoid X receptor (RXR) subtypes were analyzed by Northern blot. Immunoblot analysis of hCAR protein was also performed. The hCAR promoter's activity and its responsiveness to RA treatment was evaluated by transient transfection of the hCAR promotor-luciferase reporter constructs, followed by promoter deletion analysis to map the specific regions responsible for the RA response.

Results: In our in vitro model, both all-trans RA and 9-cis RA induced hCAR mRNA in a time- and dose-dependent manner. RA's effect was blocked by either RNA or protein synthesis inhibitors; however, hCAR mRNA's stability was not affected by RA, as determined by RNA decay experiments. Although all RAR and RXR subtypes were detected, only RXRgamma and RARalpha increased dramatically after treatment with RA. An RXR-specific agonist, but not an RAR-specific agonist, also increased hCAR mRNA and protein expression in both Weri-Rb-1 and Y79 cells. RA stimulated hCAR promoter-luciferase activity in transient transfection studies. Subsequently, a region between -852 and -702 of the hCAR promoter, with RA-responsive elements (RAREs), was discovered to be responsible for the RA response.

Conclusions: The hCAR gene is transcriptionally upregulated by RA acting through cis elements within -852 to -702 of the hCAR 5' flanking region. Based on the cumulative data, RXRgamma is most likely the RA receptor subtype involved in hCAR regulation by RA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arrestins / genetics*
  • Arrestins / metabolism
  • Base Sequence
  • Blotting, Northern
  • Dose-Response Relationship, Drug
  • Gene Expression / drug effects
  • Humans
  • Immunoblotting
  • Molecular Sequence Data
  • Plasmids
  • Promoter Regions, Genetic / genetics*
  • RNA, Messenger / metabolism
  • Receptors, Retinoic Acid / genetics
  • Receptors, Retinoic Acid / metabolism
  • Recombinant Fusion Proteins / genetics
  • Response Elements / genetics*
  • Retinal Cone Photoreceptor Cells / metabolism*
  • Retinal Neoplasms / genetics*
  • Retinal Neoplasms / metabolism
  • Retinoblastoma / genetics*
  • Retinoblastoma / metabolism
  • Retinoid X Receptors
  • Time Factors
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Tretinoin / pharmacology*
  • Tumor Cells, Cultured
  • Up-Regulation / drug effects

Substances

  • Arrestins
  • RNA, Messenger
  • Receptors, Retinoic Acid
  • Recombinant Fusion Proteins
  • Retinoid X Receptors
  • Transcription Factors
  • arrestin3
  • Tretinoin