TNFα promotes osteogenic differentiation of human mesenchymal stem cells by triggering the NF-κB signaling pathway

Abstract

Mesenchymal stem cells are multipotent cells able to differentiate into different mesenchymal lineages. Studies in the past had suggested that two of these mesenchymal differentiation directions, the chondrogenic and the myogenic differentiation, are negatively regulated by the transcription factor NF-κB. Although osteogenic differentiation has been extensively studied, the influence of NF-κB on this differentiation lineage was not subject of detailed analyses in the past. We have analyzed the consequences of TNF-α treatment and genetic manipulation of the NF-κB pathway for osteogenic differentiation of hMSCs. Treatment of hMSCs during differentiation with TNF-α activates NF-κB and this results in enhanced expression of osteogenetic proteins like bone morphogenetic protein2 (BMP-2) and alkaline phosphatase (ALP). In addition, enhanced matrix mineralization was observed. The direct contribution of the NF-κB pathway was confirmed in cells that express a constitutively active version of the NF-κB-inducing kinase IKK2 (CA-IKK2). The IKK2/NF-κB-induced BMP-2 up-regulation results in the enhancement of RUNX2 and Osterix expression, two critical regulators of the osteogenic differentiation program. Interestingly, a genetic block of the NF-κB pathway did not interfere with osteogenic differentiation. We conclude that TNFα mediated NF-κB activation, although not absolutely required for BMP-2 expression and matrix mineralization nevertheless supports osteogenic differentiation and matrix mineralization by increasing BMP-2 expression. Our results therefore suggest that NF-κB activation may function in lineage selection during differentiation of hMSCs by fostering osteogenic differentiation at the expense of other differentiation lineages.

Introduction

Adult mesenchymal stem cells (MSC) are derived from bone marrow stroma or connective tissue and can differentiate into various lineages including fibroblasts, osteoblasts, adipocytes, and chondrocytes [11], [15], [57]. The multi-lineage differentiation potential of MSC populations has been extensively studied and culturing conditions for in vitro differentiation have been established. Although much progress has been made regarding the distinct differentiation processes in the last years, the signaling pathways involved in differentiation, are not yet completely understood.

The NF-κB signaling pathway is long known to play an important role in inflammation and control of the immune system [9], [24], [47]. In addition, it regulates the transcription of genes involved in cell growth and cell death [5], [35]. NF-κB belongs to the Rel family of transcription factors and in mammals is encoded by five genes named relA, relB, c-rel, nf-κb1, and nf-κb2. All NF-κB proteins contain a conserved Rel homology domain, which is responsible for DNA binding, dimerization, and interaction with IκB proteins. In their inactive state, NF-κB proteins are located in the cytoplasm as homo- or heterodimers, bound to IκB family proteins, most importantly the IκBα protein. Upon stimulation by e.g. pro-inflammatory cytokines, such as TNF-α, the classical pathway is induced resulting in activation of the IκB-kinase-complex (IKK). This IKK-complex consists of two protein kinases (IKK1/α and IKK2/β) and a regulatory protein NEMO/IKKγ. IKK2 is largely responsible for the IκBα phosphorylation in the classical pathway. This phosphorylation triggers poly-ubiquitination and subsequent degradation of IκBα by the proteasome. NF-κB then translocates into the nucleus where it binds to specific sequences in the regulatory regions of target genes. Through a negative feedback loop newly synthesized IκBα binds to nuclear NF-κB and exports it back to the cytoplasm [9]. In addition to the classical pathway there is also an alternative pathway, which plays a central role in expression of genes involved in development and maintenance of secondary lymphoid organs. This alternative pathway is mainly stimulated via LTβR, BAFFR and CD40 leading to activation of NIK and subsequent activation of IKK1 [67].

A specific role of NF-κB in differentiation of MSC derivatives had been implicated in the past. It was shown that NF-κB induces degradation of the mRNA encoding the myogenic transcription factor MyoD in TNF-α treated myocytes [29], [60]. In addition, NF-κB is responsible for TNF-α-induced muscle protein degradation in differentiated muscle cells [41], [43], [46]. Furthermore, NF-κB influences the chondrogenic differentiation as TNF-α-induced NF-κB was shown to down-regulate mRNA levels of the chondrogenic transcription factor Sox9, thereby inhibiting differentiation of chondrocytes [53, [60]. However, there is also evidence that NF-κB can function as a positive regulator of mesenchymal cell differentiation as it was shown that NF-κB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity [66].

Less is known about the role of NF-κB signaling in osteoblast differentiation. A negative regulation of osteoblast differentiation by NF-κB was suggested as inhibition of NF-κB signaling activity in osteosarcoma cells (Saos2) results in induction of several osteogenic markers, like BMP-4 and 7, Cbfa1, alkaline phosphatase, osteopontin and osteocalcin [4]. This negative role of NF-κB was also demonstrated by the H₂O₂-induced, NF-κB-dependent reduction of osteogenic differentiation markers, like alkaline phosphatase, collagen I, and Cbfa1 in rabbit bone marrow stromal cells and calvarial osteoblasts [6]. However, NF-κB was not analyzed directly in these cases, but rather the consequences of stimuli were studied, which amongst other pathways also induce NF-κB. Indeed some inhibiting effects of TNF-α on differentiation processes were documented to be independent of NF-κB signaling. Furthermore, previous studies demonstrated that TNF-α-induced inhibition of the terminal adipogenic differentiation of pre-adipocytes [68] and inhibition of osteogenic differentiation of pre-osteoblastic cells [25], [49] is NF-κB-independent.

To elucidate the role of NF-κB in osteogenesis, we have analyzed its influence on osteogenic differentiation by infecting human mesenchymal stem cells with different NF-κB modulators. Our findings indicate that enhanced NF-κB activity in human mesenchymal stem cells increases osteogenic differentiation, whereas decreased NF-κB signaling does not impede the osteogenic differentiation.