Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. METHODS: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. RESULTS: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. CONCLUSIONS: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

Regulation of H19 and its encoded microRNA-675 in osteoarthritis and under anabolic and catabolic in vitro conditions.

Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1ß and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens.

BACKGROUND: Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases. METHODOLOGY/PRINCIPAL FINDINGS: Using patient skin biopsies, cell culture and murine infection models, HIF-1 activation was determined by immunohistochemistry, immunoblotting and reporter gene assays and was linked to cellular oxygen consumption. The course of a S. aureus peritonitis was determined upon pharmacological HIF-1 inhibition. Activation of HIF-1 was detectable (i) in all ex vivo in biopsies of patients suffering from skin infections, (ii) in vitro using cell culture infection models and (iii) in vivo using murine intravenous and peritoneal S. aureus infection models. HIF-1 activation by human pathogens was induced by oxygen-dependent mechanisms. Small colony variants (SCVs) of S. aureus known to cause chronic infections did not result in cellular hypoxia nor in HIF-1 activation. Pharmaceutical inhibition of HIF-1 activation resulted in increased survival rates of mice suffering from a S. aureus peritonitis. CONCLUSIONS/SIGNIFICANCE: Activation of HIF-1 is a general phenomenon in infections with human pathogenic bacteria, viruses, fungi and protozoa. HIF-1-regulated pathways might be an attractive target to modulate the course of life-threatening infections.

Septal and lateral wall localization of PBP5, the major D,D-carboxypeptidase of Escherichia coli, requires substrate recognition and membrane attachment.

The distribution of PBP5, the major D,D-carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild-type cells and in mutants lacking one or more D,D-carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane-bound form localized to the developing septum and restored wild-type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.