Faculty Bibliography

The urokinase-type plasminogen activator receptor (uPAR) has a well-established role in cancer progression, but it has been little studied at earlier stages of cancer initiation. Here, we show that uPAR deficiency in the mouse dramatically reduces susceptibility to the classical two-stage protocol of inflammatory skin carcinogenesis. uPAR genetic deficiency decreased papilloma formation and accelerated keratinocyte differentiation, effects mediated by Notch1 hyperactivation. Notably, Notch1 inhibition in uPAR-deficient mice rescued their susceptibility to skin carcinogenesis. Clinically, we found that human differentiated keratoacanthomas expressed low levels of uPAR and high levels of activated Notch1, with opposite effects in proliferating tumors, confirming the relevance of the observations in mice. Furthermore, we found that TACE-dependent activation of Notch1 in basal kerantinocytes was modulated by uPAR. Mechanistically, uPAR sequestered TACE within lipid rafts to prevent Notch1 activation, thereby promoting cell proliferation and tumor formation. Given that uPAR signaling is nonessential for normal epidermal homeostasis, our results argue that uPAR may present a promising disease-specific target for preventing skin cancer development. Cancer Res; 75(22); 1-15. (c)2015 AACR.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and up-regulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 microg/ml (EC50 0.5-1 mug/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/beta-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by up-regulating MMP-13 through canonical Wnt signaling.-Attur, M., Yang, Q., Shimada, K., Tachida, Y., Nagase, H., Mignatti, P., Statman, L., Palmer, G., Thorsten, K., Beier, F., Abramson, A. B. Elevated expression of periostin in human osteoarthritic cartilage and its potential role in matrix degradation via matrix metalloproteinase-13.

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non-proteolytic interactions with extracellular, intracellular, and transmembrane proteins. Here we show that in tumor cells MT1-MMP downregulates fibroblast growth factor-2 (FGF-2) signaling by reducing the amount of FGF-2 bound to the cell surface with high and low affinity. FGF-2 induces weaker activation of ERK1/2 MAP kinase in MT1-MMP expressing cells than in cells devoid of MT1-MMP. This effect is abolished in cells that express proteolytically inactive MT1-MMP but persists in cells expressing MT1-MMP mutants devoid of hemopexin-like or cytoplasmic domain, showing that FGF-2 signaling is downregulated by MT1-MMP proteolytic activity. MT1-MMP expression results in downregulation of FGFR-1 and -4, and in decreased amount of cell surface-associated FGF-2. In addition, MT1-MMP strongly reduces the amount of FGF-2 bound to the cell surface with low affinity. Because FGF-2 association with low-affinity binding sites is a prerequisite for binding to its high-affinity receptors, downregulation of low-affinity binding to the cell surface results in decreased FGF-2 signaling. Consistent with this conclusion, FGF-2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1- MMP than in MT1-MMP expressing cells. Thus, MT1-MMP controls FGF-2 signaling by a proteolytic mechanism that decreases the cell's biological response to FGF-2. J. Cell. Physiol. 230: 366-377, 2015. (c) 2014 Wiley Periodicals, Inc.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades a variety of extracellular matrix (ECM) components. In addition, MT1-MMP activates intracellular signaling through proteolysis-dependent and independent mechanisms. We have previously shown that binding of tissue inhibitor of metalloproteinases-2 (TIMP-2) to MT1-MMP controls cell proliferation and migration, as well as tumor growth in vivo by activating the Ras-extracellular signal regulated kinase-1 and -2 (ERK1/2) pathway through a mechanism that requires the cytoplasmic but not the proteolytic domain of MT1-MMP. Here we show that in MT1-MMP expressing cells TIMP-2 also induces rapid and sustained activation of AKT in a dose- and time-dependent manner and by a mechanism independent of the proteolytic activity of MT1-MMP. Fibroblast growth factor receptor-1 mediates TIMP-2 induction of ERK1/2 but not of AKT activation; however, Ras activation is necessary to transduce the TIMP-2-activated signal to both the ERK1/2 and AKT pathways. ERK1/2 and AKT activation by TIMP-2 binding to MT1-MMP protects tumor cells from apoptosis induced by serum starvation. Conversely, TIMP-2 upregulates apoptosis induced by three-dimensional type I collagen in epithelial cancer cells. Thus, TIMP-2 interaction with MT1-MMP provides tumor cells with either pro- or anti-apoptotic signaling depending on the extracellular environment and apoptotic stimulus.

Protein kinase C epsilon (PKCepsilon) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCepsilon on FGF-2 dependent vascular growth and endothelial activation. psiepsilonRACK, a selective PKCepsilon agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCepsilon in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. psiepsilonRACK fails to elicit responses in FGF-2(-/-) endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the psiepsilonRACK activity. In vivo psiepsilonRACK does not induce angiogenesis in the rabbit cornea. However, psiepsilonRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCepsilon stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.

TGF-beta1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-beta1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-beta1 induces endothelial cell expression of VEGF, which mediates TGF-beta1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-beta1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -alpha, beta, gamma, and delta-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-beta1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38alpha, beta, and gamma. VEGF activates p38beta, whereas TGF-beta1 activates p38alpha. TGF-beta1 treatment rapidly induces p38alpha activation and apoptosis. Subsequently, p38alpha activation is downregulated, p38beta is activated, and the surviving cells become refractory to TGF-beta1 induction of apoptosis and proliferate. Gene silencing of p38alpha blocks TGF-beta1 induction of apoptosis, whereas downregulation of p38beta or p38gamma expression results in massive apoptosis. Thus, in endothelial cells p38alpha mediates apoptotic signaling, whereas p38beta and p38gamma transduce survival signaling. TGF-beta1 activation of p38alpha is mediated by VEGF, which in the absence of TGF-beta1 activates p38beta. Therefore, these results show that TGF-beta1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38beta to the proapoptotic p38alpha. Mol Cancer Res; 10(5); 605-14. (c)2012 AACR.

Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF), both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-beta1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-beta1 induces endothelial cell expression of VEGF, which mediates TGF-beta1 induction of apoptosis. VEGF occurs in several isoforms derived from alternative splicing of a single gene. A recently described family of isoforms, called b, is generated through alternative splice-site selection in the last exon. Therefore, VEGFb isoforms carry 6 C-terminal amino acids different from the other VEGF isoforms. It has been reported that VEGFb isoforms inhibit angiogenesis by competing with VEGF for receptor binding. Based on previous findings that TGF-beta1 upregulates VEGFb expression in renal cells, we hypothesized that TGF-beta1 induces VEGFb expression in vascular endothelial cells, and that VEGFb induces endothelial cell apoptosis. To test our hypothesis we treated human or bovine endothelial cells with TGF-beta1 (1 ng/ml) for 6 h, a time sufficient for apoptosis induction, and characterized VEGFb expression and MAP kinase activation by Western blotting. The results showed that TGF-beta1 induced VEGFb expression. Treatment of endothelial cells with pure, recombinant VEGFb rapidly activated p38MAPK and induced apoptosis. Similarly, transfection of endothelial cells with VEGFb cDNA resulted in increased apoptosis. In contrast, purified VEGF or transfection with VEGF cDNA protected the cells from apoptosis. Therefore, our data show that TGF-beta1 induces endothelial cell apoptosis by upregulating expression of VEGFb, which relays apoptotic signaling through an autocrine mechanism. A number of tissues, particularly tumors, express high levels of VEGF. The pharmacological control of VEGFb isoform expression could be exploited to induce endothelial cell apoptosis and thus block angiogenesis

Transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF), both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-beta1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-beta1 induces endothelial cell expression of VEGF, which mediates TGF-beta1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38MAPK but protects the cells from apoptosis, this finding suggested that TGF-beta1 converts p38MAPK signaling from prosurvival to proapoptotic. Four isoforms of p38MA