The orphan nuclear receptor, ROR/RZR, has no known natural ligand. Studies to date with ROR[alpha] gene knockout in mice and with naturally occuring mutantions/deletions in the ligand binding domain (LBD)/ E region demonstrate a role for ROR[alpha] in:
• Purkinje cell development;
• cerebellar ataxia;
• lipid metabolism;
• immune response.
The mRNA encoding ROR[alpha]1 is abundantly expressed in skeletal muscle. However, the role ofthe orphan nuclear receptor, ROR[alpha]1, in the process of muscle differentiation remains unclear.
Exogenous expression of dominant negative ROR[alpha]1 (1-235) mRNA (which lacks the ligand binding domain) in myogenic cells significantly reduces the endogenous expression of ROR[alpha]1 mRNA, represses the accumulation and delays the activation of mRNAs encoding myoD, myogenin [the muscle-specific basic helix-loop-helix (bHLH) proteins] and p21Waf-l/Cip-l (a cdk inhibitor). Immunohistochemistry demonstrates that morphological differentiation (assayed by the accumulation of the major thick filament protein, MHC) is delayed in cells expressing the ROR[alpha]1(l-235) transcript. Furthermore, the size and development of multinucleated myotubes is impaired. A similar inhibition of gene expression can be found when antisense ROR[alpha]1 RNA is expressed. In contrast, when ROR[alpha]1 is stably expressed, p21 and a-actin transcription is induced. This suggests that ROR[alpha]1 potentiates skeletal muscle differentiation.
Constitutive transactivation is the key function of ROR[alpha]1. GAL4 hybrid assay revealed that transactivation required the contribution of both the hinge/D region as well as the LBD/E region of ROR[alpha]1. The LBD fragment alone would not function, reinforcing the necessity of the hinge region. Surprisingly, the hinge region independently activates transcription. This activity is encoded by the more distal region of the hinge fragment and may be potentiated by p300. We also showed that ROR[alpha]1 is transcriptionally active without external stimulus confirming ROR[alpha]1 is an orphan nuclear receptor. Non-lipophilic serum factors potentiate ROR[alpha]1 transactivational activity. This activity however, is not due to activation by cAMP.
ROR[alpha]1 interacts with coactivator p300 in vitro and in vivo, a cofactor that coactivates nuclear receptor and myoD-mediated transactivation. In vitro GST-pulldown showed that ROR[alpha]1 can interact with N-CoR and its variants, however, this interaction did not occur in a cellular context. The E region of ROR[alpha]1 interacts with p300. Consistent with the positive role of ROR[alpha]1 in myogenesis, we observed that ROR[alpha]1 directly interacts with myoD. This interaction was mediated by the N-terminal activation domain of myoD, and the ROR[alpha]1 DNA binding domain/C region. Furthermore, we demonstrated that p300, ROR[alpha]1 and myoD interact in a non-competitive manner. In conclusion, this study provides evidence for a biological role for ROR[alpha]1 with positive influence in the cascade of events involved in the activation of myogenic-specific markers and cell cycle regulators and suggests that cross-talk between the ROR[alpha]1 and the myogenic bHLH proteins has functional consequences for differentiation.