Ets domain transcription factor PE1 suppresses human interstitial collagenase promoter activity by antagonizing protein - DNA interactions at a critical AP1 element

In MC3T3E1 calvarial osteoblasts, fibroblast growth factor receptor (FGFR) signaling elicits multiple transcriptional responses, including upregulatio n of the interstitial collagenase/matrix metallo-proteinase 1 (MMPI) promot er. FGF responsiveness maps to a bipartite Ets/AP1 element at base pairs -1 23 to -61 in the human MMPI promoter. Under basal conditions, the MMP1 prom oter is repressed in part via protein-DNA interactions at the Ets cognate, and minimally two mechanisms convey MMP1 promoter upregulation by FGF2: (a) transcriptional activation via Fra1/c-Jun containing DNA-protein interacti ons at the API cognate and (b) derepression of promoter activity regulated by the Ets cognate. To identify osteoblast Ets repressors that potentially participate in gene expression in the osteoblast, we performed reverse tran scription-polymerase chain reaction (RT-PCR) analysis of mRNA isolated from MC3T3E1 cells, using degenerative amplimers to the conserved Ets DNA bindi ng domain to survey the Ets genes expressed by these cells. Six distinct Et s mRNAs were identified: Ets2, Fli1, GABP alpha, SAP1, Elk1, and PE1, Of th ese, only PEI has extensive homology to the known Ras-regulated Ets transcr iptional repressor, ERF. Therefore, we cloned and characterized PEI cDNA fr om a mouse brain library and performed functional analysis of this particul ar Ets family member. A 2 kb transcript was isolated from brain that encode s a similar to 57 kDa protein; the predicted protein contains the known N-t erminal Ets domain of PE1 and a novel C-terminal domain with significant ho mology to murine ERF. The murine PE1 open reading Frame (ORF) is much large r than the previously reported human PE1 ORF. Consistent with this, affinit y-purified rabbit anti-mouse PEI antibody specifically recognizes an simila r to 66 kDa protein present only in the nuclear fraction of MC3T3E1 osteobl asts. Recombinant PE1 binds authentic AGGAWG Ets DNA cognates, and transien t transfection studies demonstrate that PE1 represses MMPI promoter activit y. Surprisingly, although deletion of the MMP1 Ets cognate at nucleotides - 88 to -83 abrogates FGF2 induction, it does not prevent suppression of the AP1-dependent MMP1 promoter by PE1. PE1 regulation maps to the MMPI promote r region -75 to -61, suggesting that PE1 suppresses transcription via prote in-protein interactions with AP1. Consistent with this, recombinant GST-PE1 specifically inhibits the formation of protein-DNA interactions on the MMP 1 AP1 site (-72 to -66) when present in an admixture with MC3T3E1 crude nuc lear extract. In tote, these data indicate that PE1 participates in the tra nscriptional regulation of the MMP1 promoter in osteoblasts. As observed wi th other transcriptional repressors of MMP1 gene expression, transcriptiona l suppression by PE1 occurs via inhibition of AP1-dependent promoter activi ty.