In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage. IntroductionOxygen is essential to life for all higher organisms. Molecular oxygen is required as an electron acceptor in the generation of cellular energy (ATP) through the process of oxidative phosphorylation, and it is also used as a substrate in various enzymatic reactions [1]. Oxygen homeostasis is, therefore, a basic requirement and complex systems have evolved to maintain this at the cell, tissue and whole organism levels. These include increased reliance on anaerobic glycolysis in the formation of ATP within the cell; increased angiogenesis and blood supply (through vasodilation) to affected organs; and systemic changes such as enhanced erythropoiesis and increased ventilation [2,3].Cartilage develops in a hypoxic environment [4], and indeed proximity to a blood supply appears to be a determining factor in the formation of bone over cartilage [5,6]. In addition, due to the absence of vasculature, articular cartilage (unlike most tissues) is maintained and functions in a low oxygen environment throughout life [7][8][9][10]. The resident cells, the chondrocytes, are the only cell type present in the tissue and appear to have developed specific mechanisms to promote tissue function in response to this chronic hypoxia, for example, by inducing increased expression of cartilage matrix components [11][12][13], and through the inhibition of angiogenesis [14]. In addition to mediating the ubiquitous hypoxia responses, hypoxia-inducible factors (HIFs) also appear to be critical to these tissue-specific responses in chondrocytes. Hypoxia-inducible factorsIn the mid-1990s a major breakthrough was made in our understanding of the molecular mechanisms mediating cellular responses to hypoxia with the discovery of HIF-1 [15]. The stability and function of HIF is regulated post-translationally by hydroxylation of specific amino acid residues. In the presence of sufficient molecular oxygen, HIF is degraded almost as soon as it is made due to hydroxylation of specific proline residues that target the HIF-α subunit for Von HippelLindau tumour suppressor protein (pVHL)-mediated proteosomal degradation. Conversely, when oxygen levels are limiting (typically <5%), hydroxylation is inhibited and HIF-α escapes degradation, and is free to heterodimerise with the constitutively expressed HIF-β subunit (also called Aryl hydrocarbon nuclear translocator (ARNT)). This complex translocates to the nucleus, binding specific consensus seq...

Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein–DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 μM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3α and STAT3β. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression.

Mesenchymal stem cells (MSCs) have great potential for cell-based therapies. However, lack of cell-specific markers thwarts full realization of this as it prevents their identification in vivo, and subsequent purification. In the present study, to ensure cell purity multiple individual clones were derived from the bone marrow of BALB/b and BALB/c mice, and subsequently defined as MSCs by demonstrating their multipotentiality and self-renewal ability. In an effort to define the molecular signature of such MSCs and identify potentially cell-specific markers, an extensive genome-wide microarray analysis was performed comparing eight individual undifferentiated MSC clones to four different controls-corresponding differentiated MSC clones, bone marrow adherent cells, freshly isolated bone marrow cells, and embryonic fibroblasts. Strikingly, all MSC clones expressed differentially high levels of six-transmembrane epithelial antigen of the prostate (STEAP1 and STEAP2). Further, both STEAP members showed an extremely similar expression profile to stem cell antigen-1 (Sca-1) as demonstrated by two-dimensional hierarchical cluster analysis. Most importantly, differentially high levels of STEAP1 and STEAP2 proteins were also detected in human multipotent bone marrow adherent cultures. Thus, STEAPs may represent novel markers of MSCs in man as well as mice. Depletion of STEAP1 in human MSCs using RNAi resulted in decreased cell adhesion to tissue culture plastic. Further work is now needed to fully uncover its function in these cells, and to explore its potential as a marker of MSCs.

No abstract