Activation of mechanosensitive ion channel TRPV4 normalizes tumor vasculature and improves cancer therapy.

Tumor vessels are characterized by abnormal morphology and hyperpermeability that together cause inefficient delivery of chemotherapeutic agents. Although vascular endothelial growth factor has been established as a critical regulator of tumor angiogenesis, the role of mechanical signaling in the regulation of tumor vasculature or tumor endothelial cell (TEC) function is not known. Here we show that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) regulates tumor angiogenesis and tumor vessel maturation via modulation of TEC mechanosensitivity. We found that TECs exhibit reduced TRPV4 expression and function, which is correlated with aberrant mechanosensitivity towards extracellular matrix stiffness, increased migration and abnormal angiogenesis by TEC. Further, syngeneic tumor experiments revealed that the absence of TRPV4 induced increased vascular density, vessel diameter and reduced pericyte coverage resulting in enhanced tumor growth in TRPV4 knockout mice. Importantly, overexpression or pharmacological activation of TRPV4 restored aberrant TEC mechanosensitivity, migration and normalized abnormal angiogenesis in vitro by modulating Rho activity. Finally, a small molecule activator of TRPV4, GSK1016790A, in combination with anticancer drug cisplatin, significantly reduced tumor growth in wild-type mice by inducing vessel maturation. Our findings demonstrate TRPV4 channels to be critical regulators of tumor angiogenesis and represent a novel target for anti-angiogenic and vascular normalization therapies.

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells.

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.

Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells.

The proinflammatory mediator leukotriene D(4) (LTD(4)) binds to the seven-transmembrane receptor CYSLT(1). Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD(4) induced tyrosine phosphorylation and translocation of phospholipase (PLC)-gamma1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD(4) induced a rapid interaction between Gbetagamma subunits and PLC-gamma1; results obtained with GST fusion proteins of PLC-gamma1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-gamma1. Moreover, LTD(4) induced an increased association of c-Src with PLC-gamma1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD(4)-induced tyrosine phosphorylation of PLC-gamma1 and the association of PLC-gamma1 with Gbetagamma subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gbeta, anti-PLC-gamma1, or anti-c-Src antibodies and by pretreatment with PP1. LTD(4)-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gbetagamma subunits can, directly or indirectly, serve as membrane-bound partners for PLC-gamma1 and c-Src and that each of these proteins is essential for LTD(4)-induced downstream PLC-gamma1 signaling.

Leukotriene D4 induces a rapid increase in cAMP in the human epithelial cell line, Int 407: a potential role for this signal in the regulation of calcium influx through the plasma membrane.

Although the LTD4-induced Ca2+ influx in human epithelial cells has been shown to be regulated by a pertussis toxin-sensitive heterotrimeric G-protein, most likely a G alpha i3 protein [Adolfsson J.L.P., Ohd J.F., Sjölander A. Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells. Biochem Biophys Res Commun 1996; 226: 413-419], the signalling pathway further downstream is still unclear. In the present study, we investigated the possible involvement of cAMP and protein kinase A activity in the LTD4-induced Ca2+ influx in the epithelial cell line Int 407. Stimulation with LTD4, but not with the calcium ionophore ionomycin, triggered a rapid increase (peak at 7 s) in the cellular cAMP level, an effect that was totally abolished by pertussis toxin. Furthermore, the LTD4-induced Ca2+ signal was reduced by 60% when cells that had been pre-incubated with the protein kinase A inhibitor Rp-cAMPS (50 microM for 30 min) were stimulated in a calcium containing medium. In contrast, Rp-cAMPS had no apparent effect on the LTD4-induced Ca2+ signal when the cells were stimulated in a calcium-depleted medium. The immediate LTD4-induced protein tyrosine phosphorylation (15 s), previously shown to be necessary for the subsequent Ca2+ influx, was abolished not only by pretreatment with pertussis toxin but also by exposure to Rp-cAMPS. Furthermore, direct activation of the cellular adenylyl cyclase activity by treatment with forskolin alone induced a prompt Ca2+ signal in the presence, but not in the absence, of extracellular Ca2+, identical results were obtained with the cell permeable cAMP analogue 8-bromo-cAMP. In addition, forskolin induced protein tyrosine phosphorylation similar to that seen with LTD4. These results suggest that protein kinase A activity participates in the regulation of the LTD4-induced Ca2+ influx at a site that is downstream of the activation of the pertussis toxin-sensitive G-protein but upstream of a LTD4-stimulated tyrosine kinase(s).