NF-κB suppresses HIF-1α response by competing for P300 binding

Abstract

Hypoxia has emerged as a key determinant of osteogenesis. HIF-1α is the transcription factor mediating hypoxia responses that include induction of VEGF and related bone induction. Inflammatory signals antagonize bone repair via the NF-κB pathway. The present investigation explored the functional relationship of hypoxia (HIF-1α function) and inflammatory signaling (NF-κB) in stem like and osteoprogenitor cell lines. The potential interaction between HIF-1α and NF-κB signaling was explored by co-transfection studies in hFOB with p65, HIF-1α and 9x-HRE-luc or HIF-1α target genes reporter plasmids. Nuclear cross-talk was directly tested using the mammalian Gal4/VP16 two-hybrid, and confirmed by co-immunoprecipitation/western blotting assays. The results show that inflammatory stimulation (TNF-α treatment) causes a marked inhibition of HIF-1α function at the HRE in all cell lines studied. Also, co-transfection with p65 expression vector leads to reduced hVEGFp transcription after DFO-induced hypoxia. However, TNF-α treatment had little effect on HIF-1α mRNA levels. The functional interaction of Gal4-HIF-1α and VP16-p300 fusion proteins is effectively blocked by expression of p65 in a dose dependent manner. It was concluded that NF-κB-mediated inflammatory signaling is able to block HIF-1α transactivation at HRE-encoding genes by direct competition for p300 binding at the promoter. Inflammation may influence the stem cell niche and tissue regeneration by influencing cellular responses to hypoxia.

Introduction

Bone formation and repair is dependent on the recruitment and differentiation of mesenchymal stem cells (or osteoprogenitor cells) in spatial and lineage specific ways. The many well orchestrated biological steps involve transcriptional regulation of the mesenchymal stem cell. Multiple environmental signals converge at the site of formation and repair within a systemic biologic context to establish a cell population capable of osteogenesis. Within bone marrow, hypoxia has emerged as one of the key determinants of this specific signaling of osteogenesis. The role of hypoxia on osteogenesis is clearly illustrated by deletion of the von Hippel-Lindau gene (VHL) that regulates cellular responses to hypoxia by the Hypoxia-Inducible Factor (HIF) pathway. In the absence of VHL, mice develop extremely dense, highly vascularized long bones. Suggested is a critical link between hypoxia, vascularization and osteogenesis [1].

HIF-1 is a heterodimeric transcription factor that is composed of a constitutively expressed HIF-1β subunit (also know as ARNT) and an oxygen-regulated HIF-1α subunit. HIF-1α abundance is controlled by ubiquitination and proteasomal degradation. During normoxia, HIF-1α is continuously synthesized and degraded, and degradation is triggered by binding of the von Hippel-Lindau tumor-suppressor protein (VHL), and posterior degradation by the 26S proteasome. Hypoxia results in HIF-1α stabilization, nuclear translocation and transcription of genes containing hypoxia response elements (HRE (5′-RCGTG-3′)). Bound HIF-1α/HIF-1β interacts with the coactivator protein p300 that results in transactivation at hypoxia responsive genes [2], [3]. The VEGF gene promoter exemplifies that this regulation and this hypoxia-mediated mechanism is central to the observations made concerning VHL^−/− as well as HIF-1α^−/− mice.

Bone repair is antagonized by inflammation. Several different investigations have shown that inflammatory stimuli reduce or preclude bone repair or bone formation. For example, TNF-α treatment of osteoprogenitor cells blocks induced osteoblast differentiation. One possible mechanism is the TNF-α mediated antagonism of bone morphogenetic protein signaling [4]. Another possible effect of inflammatory signaling in osteoblastic cells is the direct inhibition of key transcriptional regulators such as Osterix [5], [6]. Although HIF-1α has a well defined role in positive regulation of vascularization through transactivation of VEGF and is further considered important in the process of wound repair, initial studies of inflammatory modulation of osteoblastic differentiation suggested that inflammatory signaling strongly antagonized HIF-1α function in osteoprogenitor cells. The aim of this investigation was to investigate how inflammatory signaling inhibited HIF-1α function in different cell lines. These studies demonstrate that inflammatory signaling mediated by NF-κB is able to block transactivation at HRE-encoding genes by HIF-1α by direct competition for p300 binding at the promoter.