The LIM domain protein FHL2 interacts with the NR5A family of nuclear receptors and CREB to activate the inhibin-α subunit gene in ovarian granulosa cells.
Nuclear receptor transcriptional activity is enhanced by interaction with coactivators. The highly related nuclear receptor 5A (NR5A) subfamily members liver receptor homolog 1 and steroidogenic factor 1 bind to and activate several of the same genes, many of which are important for reproductive function. To better understand transcriptional activation by these nuclear receptors, we sought to identify interacting proteins that might function as coactivators. The LIM domain protein four and a half LIM domain 2 (FHL2) was identified as interacting with the NR5A receptors in a yeast two-hybrid screen of a human ovary cDNA library. FHL2, and the closely related FHL1, are both expressed in the rodent ovary and in granulosa cells. Small interfering RNA-mediated knockdown of FHL1 and FHL2 in primary mouse granulosa cells reduced expression of the NR5A target genes encoding inhibin-α and P450scc. In vitro assays confirmed the interaction between the FHL and NR5A proteins and revealed that a single LIM domain of FHL2 is sufficient for this interaction, whereas determinants in both the ligand binding domain and DNA binding domain of NR5A proteins are important. FHL2 enhances the ability of both liver receptor homolog 1 and steroidogenic factor 1 to activate the inhibin-α subunit gene promoter in granulosa cells and thus functions as a transcriptional coactivator. FHL2 also interacts with cAMP response element-binding protein and substantially augments activation of inhibin gene expression by the combination of NR5A receptors and forskolin, suggesting that FHL2 may facilitate integration of these two signals. Collectively these results identify FHL2 as a novel coactivator of NR5A nuclear receptors in ovarian granulosa cells and suggest its involvement in regulating target genes important for mammalian reproduction.
Department of Molecular Biosciences and Center of Reproductive Science, Northwestern University, Evanston, Illinois 60208, USA.
Christina K
CK
Matulis
Kelly E
KE
Mayo
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006107:Granulosa Cells / Q000378:metabolism*; D007265:Inhibins / Q000235:genetics* / Q000378:metabolism; D060850:LIM-Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D020218:Response Elements; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D054339:Steroidogenic Factor 1 / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; C491382:Fhl2 protein, mouse; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; C431936:Nr5a2 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D012269:Ribosomal Proteins; C489093:Rpl19 protein, mouse; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C094377:inhibin-alpha subunit; C515058:steroidogenic factor 1, mouse; D007265:Inhibins; D000242:Cyclic AMP