Abstract
The expression of inducible cAMP early repressor (ICER) has been demonstrated in cultured osteoblastic MC3T3-E1 cells, calvarial cultures and in vivo, mainly via the cAMP-PKA signaling pathway. To determine the molecular mechanism(s) for this action, a fragment of the CREM P2 promoter from -238 bp to +14 bp was linked to a luciferase reporter (CREMP2-Luc238). This region contains 2 clusters of cAMP-responsive element (CRE). The 5’ cluster contains CRE1 and CRE2 while the 3’ cluster contains CRE3 and CRE4 and the two clusters are separated by 12 bp. In order to study if any or all CREs are responsive to cAMP induction, osteoblastic MC3T3-E1 cells were stably transfected with constructs having serial deletions of each CRE or mutations in each cluster of the CREMP2-Luc construct. Agonists that stimulate the cAMP-PKA pathway induced CREMP2-Luc238 activity, and neither PKC nor Ca++ pathways did. The deletion of CRE1 and CRE2 did not significantly abolish the cAMP inducibility of CREMP2-Luc238 activity. Electrophoretic mobility shift assay showed many DNA/protein complexes when probes from either CRE1-2 or CRE3-4 were incubated with FSK-treated nuclear extract from MC3T3-E1 cells. However, only a small portion of the complexes was competed by a consensus CRE oligonucleotide. Interestingly, a major DNA/protein complex seen in the binding of CRE3-4 was not competed by the consensus CRE oligonucleotide, but by a mutated CRE oligonucleotide suggesting that other transcription factor(s) may be involved in the binding of CRE3-4. The CRE binding DNA/protein complexes were supershifted mainly by antibodies against ICER and CREB, mildly by C/EBPβ suggesting these transcription factors may be involved in the FSK induction of the P2 promoter of the CREM gene.
