Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. We report that Pdgfrb-Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the αv integrin subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Depletion of the αv integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of αv integrins using this system was also protective in models of pulmonary and renal fibrosis. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. These data identify a core pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Objective. Recent published studies have shown that cartilage from ADAMTS-5-knockout mice, but not ADAMTS-4-or ADAMTS-1-knockout mice, is significantly protected from degradation. The present study was undertaken to evaluate the respective roles of these enzymes in human cartilage breakdown, using a small interfering RNA (siRNA) approach to assess the effects of inhibition of each enzyme in normal and osteoarthritic (OA) explants.Methods. The activities of siRNA specifically targeting ADAMTS-1, -4, and -5 were assessed by transfection into primary human chondrocytes and cultured human cartilage explants. At 24 hours, a cytokine stimulus was applied to normal, but not OA, samples to initiate a catabolic response. At designated times, total RNA was isolated and gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction. Aggrecan release and aggrecanasegenerated neoepitope formation were determined by dye binding analysis and Western blotting, respectively.Results. Human chondrocytes and explants were efficiently transfected with siRNA that specifically decreased the expression of each targeted gene. Suppression of ADAMTS-4 and ADAMTS-5, individually or in combination, attenuated the degradation of aggrecan in cytokine-stimulated normal cartilage. A reduction in aggrecan degradation was also observed following siRNA-mediated knockdown of either gene in unstimulated OA cartilage. In contrast, knockdown of ADAMTS-1 failed to inhibit aggrecan loss.Conclusion. Despite the apparent dominant role of ADAMTS-5 in genetically modified mice, our data suggest that both ADAMTS-4 and ADAMTS-5 contribute to the structural damage that characterizes human OA.

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The angiogenic inducer CYR61 is differentially overexpressed in breast cancer cells exhibiting high levels of Heregulin (HRG), a growth factor closely associated with a metastatic breast cancer phenotype. Here, we examined whether CYR61, independently of HRG, actively regulates breast cancer cell survival and chemosensitivity, and the pathways involved. Forced expression of CYR61 in HRG-negative MCF-7 cells notably upregulated the expression of its own integrin receptor a v b 3 (>200 times). Small peptidomimetic a v b 3 integrin antagonists dramatically decreased cell viability of CYR61-overexpressing MCF-7 cells, whereas control MCF-7/V remained insensitive. Mechanistically, functional blockade of a v b 3 specifically abolished CYR6-induced hyperactivation of ERK1/ERK2 MAPK, whereas the activation status of AKT did not decrease. Moreover, CYR61 overexpression rendered MCF-7 cells significantly resistant (>10-fold) to Taxol-induced cytotoxicity. Remarkably, a v b 3 inhibition converted the CYR61-induced Taxol-resistant phenotype into a hypersensitive one. Thus, the augmentation of Taxol-induced apoptotic cell death in the presence of a v b 3 antagonists demonstrated a strong synergism as verified by the terminal transferase-mediated dUTP nickend labeling (TUNEL) assay and by flow cytometric analysis for DNA content. Indeed, functional blockade of a v b 3 , similarly to the pharmacological MAPK inhibitor U0126, synergistically increased both the proportion of CYR61-overexpressing breast cancer cells in the G 2 phase of the cell cycle and the appearance of sub-G 1 hypodiploid (apoptotic) cells caused by Taxol. Strikingly, CYR61 overexpression impaired the accumulation of wild-type p53 following Taxol exposure, while inhibition of a v b 3 or ERK1/ERK2 MAPK signalings completely restored Taxol-induced upregulation of p53. Moreover, antisense downregulation of CYR61 expression abolished the anchorage-independent growth of breast cancer cells engineered to overexpress HRG, and significantly increased their sensitivity to Taxol. Our data provide evidence that CYR61 is sufficient to promote breast cancer cell proliferation, cell survival, and Taxol resistance through a a v b 3 -activated ERK1/ERK2 MAPK signaling. The identification of a 'CYR61-a v b 3 autocrine loop' in the epithelial compartment of breast carcinoma strongly suggests that targeting a v b 3 may simultaneously prevent breast cancer angiogenesis, growth, and chemo resistance.

Glucose (catabolite) repression is mediated by multiple mechanisms that combine to regulate transcription of the GAL genes over at least a thousandfold range. We have determined that this is due predominantly to modest glucose repression (4-to 7-fold) of expression of GAL4, the gene encoding the transcriptional activator of the GAL genes. GALA regulation is affected by mutations in several genes previously implicated in the glucose repression pathway; it is not dependent on GAL4 or GAL80 protein function. GALA promoter sequences that mediate glucose repression were found to lie downstream of positively acting elements that may be "TATA boxes." Two nearly identical sequences (10/12 base pairs) in this region that may be binding sites for the MIG1 protein were identified as functional glucose-control elements. A 4-base-pair insertion in one of these sites causes constitutive GAL4 synthesis and leads to substantial relief (50-fold) of glucose repression ofGAL] expression. Furthermore, promoter deletions that modestly reduce GALA expression, and therefore presumably the amount of GAL4 protein synthesized, cause much greater reductions in GAL] expression. These results suggest that GALA works synergistically to activate GALl expression. Thus, glucose repression of GAL) expression is due largely to a relatively small reduction of GALA protein levels caused by reduced GALA transcription. This illustrates how modest regulation of a weakly expressed regulatory gene can act as a sensitive genetic switch to produce greatly amplifiled responses to environmental changes.Expression of the GAL], -7, and -10 genes, which are required for galactose catabolism in Saccharomyces cerevisiae, is regulated at two levels (1). (i) Galactose induces their transcription by preventing GAL80 protein from inhibiting function of the GAL4 transcriptional activator. (ii) Glucose causes severe repression of GAL gene transcription by a process to which several different mechanisms contribute. Some operate to reduce the amount of inducer available to inactivate GAL80 by reducing expression of GAL3, required for inducer synthesis (2), and of GAL2, encoding the galactose transporter (3), and by inactivation of preexisting galactose permease in the cell (4). Other mechanisms of repression operate through sites in the GAL promoters termed the upstream activation sequence (UAS) and the upstream repression sequence (URS) and, therefore, act more directly to repress transcription.The UAS and URS regions from the GAL] promoter are capable of independently mediating glucose repression (5). The repression that operates through the UAS region, which contains four binding sites for the GAL4 activator, probably reflects reduced levels or reduced function of the GAL4 protein in glucose-grown cells. Repression mediated by the URS, which lies between the UAS and the "TATA element," is presumably due to unidentified repressors that bind to this region.UAS-mediated repression is characterized by the failure of GAL4 to bind the UAS in cells growing in the pr...

The involvement of a v h 3 and a v h 5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of a v h 3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates a v h 3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 Â 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of a v h 3 integrin when tumors protect their microvasculature from radiation-induced damage.The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.

Summary The epidermal growth factor receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis after injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functioned by releasing bioactive transforming growth factor beta (TGF-β) from latent complexes via integrin-α V activation. Using acute injury models in two different tissues, we found that by inducing TGF-β activation on mesenchymal stromal cells (pericytes), Amphiregulin induced their differentiation into myofibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration after acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

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