HES1 Cooperates With pRb to Activate RUNX2-Dependent Transcription

Abstract

The retinoblastoma protein, pRb, can activate the transcription factor RUNX2, an essential regulator of osteogenic differentiation, but the mechanism of this activation is unknown. Here we studied the interaction of pRb and RUNX2 with HES1, previously reported to augment RUNX2 activity. PRb can act to promote RUNX2/HES1 association with concomitant promoter occupancy and transcriptional activation in bone cells.Introduction: RUNX2 (also known as OSF2/CBFA1) is a transcription factor required for osteoblast differentiation and bone formation. We have reported that RUNX2 can associate with the retinoblastoma protein pRb, a common tumor suppressor in bone, and the resultant complex can bind and activate transcription from bone-specific promoters. This activity of the pRb/RUNX2 complex may thus link differentiation control with tumor suppressor activity. However, the mechanism through which pRb can activate RUNX2 is unknown. HES1 is a reported co-activator of RUNX2 that shares a binding site on RUNX2 with pRb. Thus, we have tested the cooperativity among these factors in activating transcription from bone specific promoters. Materials and Methods: Coimmunoprecipitation, chromatin immunoprecipitation, and EMSA experiments were used to study the interaction of RUNX2, HES1, and pRb in cell lysates and on DNA. Transcriptional reporter assays were used to analyze the activity of RUNX2 in the presence and absence of HES1 and pRb. Results: We showed that pRb can associate with HES1, a previously described RUNX2 interactor that can itself augment RUNX2-dependent transcription. The association of HES1 with RUNX2 is augmented by pRb. Furthermore, both pRb and HES1 increase the amount of RUNX2 bound to promoter sites in vivo, pRb and HES1 synergistically activate a RUNX2-dependent reporter gene, and depletion of HES1 reduces RUNX2/ pRb activity. Conclusions: These data indicate that pRb acts as a RUNX2 co-activator at least in part by recruiting HES1 into the pRb/RUNX2 complex and further elucidate a novel role for pRb as a transcriptional co-activator in osteogenesis.

Inhibition of Notch1 signaling by Runx2 during osteoblast differentiation

“…On the contrary, Lee and colleagues recently reported that Hes1, which is another downstream transducer of Notch1 signaling, stimulated Runx2 activity by cooperating with retinoblastoma protein (pRb). (51) Furthermore, Jung and colleagues reported that Hes1 overexpression led to an increase in osteoblast differentiation, presumably resulting from enhanced Runx2 activity elevating Runx2 target gene expression. From cDNA microarray data it was shown that Notch1, Notch3, and NOV genes are downregulated but Hey1 is upregulated during osteoblast differentiation of MC3T3 cells, suggesting that Hey1 is a BMP-2-induced gene through a Notch1-independent manner.…”

Hes1: a key role in stemness, metastasis and multidrug resistance

“…41 Additionally, Hes1 increases Runx2-DNA binding, stimulates Runx2 transcriptional activity by increasing the stability of Runx2, and associates with pRb synergistically to augment Runx2 transcription. 42,43 When examined in prostate tumor bone metastasis, Runx2 DNA-binding activity is enhanced in response to osteogenic induction and is inhibited by Notch-Hes1 inhibition, verifying that Hes1-Runx2 signaling functions critically in tumor bone metastasis. 44 Furthermore, EMT process allows the loss of junction with adjacent cells, the polarization of epithelial cells and subsequent acquisition of mesenchymal features.…”

“…Hes1 amplifies Runx2 expression by cooperating with pRb, and WRPW domain is necessary for Hes1-pRb interacion. 42 In addition, Hes1 uses bHLH domain and orange domain to bind with STAT3 and activates STAT3 phosphorylation. 58 The role of Hes1 in CSCs maintenance Hes1 is widely expressed in different tissue and cell types, including neuronal stem cells, embryonic stem cells, quiescent cells, and especially in cells at the precursor stage.…”

Functional relevance of the BMD-associated polymorphism rs312009: Novel Involvement of RUNX2 in LRP5 transcriptional regulation

“…(9) Among the cofactors that interact with Runx proteins are coactivators such as p300 and CREB-binding protein (CEBP) and co-repressors such as mSin3A, transducin-like enhancer of split proteins (TLEs), and several histone desacetylases (Hdacs). Several RUNX2 responsive elements have been identified in important bone-related genes, such as osteocalcin, (10)(11)(12)(13)(14)(15)(16) osteoprotegerin, (17) osterix, (18) and bone sialoprotein, (19)(20)(21) among others. More interestingly, functional RUNX2 binding sites have been described in the promoters of genes of other Wnt signaling pathway elements, such as SOST (22) and AXIN2 genes.…”