Sodium-proton exchanger type 1 (NHE-1) is ubiquitously expressed, is activated by numerous growth factors, and plays significant roles in regulating intracellular pH and cellular volume, proliferation and cytoskeleton. Despite its importance, little is known about its regulation in renal glomerular podocytes. In the current work, we studied the regulation of NHE-1 activity by the epidermal growth factor receptor (EGFR) in cultured podocytes. RT-PCR demonstrated mRNAs for NHE-1 and NHE-2 in differentiated podocytes, as well as for EGFR subunits EGFR/ErbB1, Erb3, and ErbB4. EGF induced concentration-dependent increases in proton efflux in renal podocytes as assessed using a Cytosensor microphysiometer, were diminished in the presence of 5-(N-methyl-N-isobutyl) amiloride or in a sodium-free solution. Furthermore, pharmacological inhibitors of janus kinase (Jak2) and calmodulin (CaM) attenuated EGF-induced NHE-1 activity. Co-immunoprecipitation studies determined that EGF induced formation of complexes between Jak2 and CaM, as well as between CaM and NHE-1. In addition, EGF increased levels of tyrosine phosphorylation of Jak2 and CaM. The EGFR kinase inhibitor, AG1478, blocked activation of NHE-1, but did not block EGF-induced phosphorylation of Jak2 or CaM. These results suggest that EGF induces NHE-1 activity in podocytes through two pathways: (1) EGF → EGFR → Jak2 activation (independent of EGFR tyrosine kinase activity) → tyrosine phosphorylation of CaM → CaM binding to NHE-1 → conformational change of NHE-1 → activation of NHE-1; and (2) EGF →EGFR → EGFR kinase activation → association of CaM with NHE-1 (independent of Jak2) → conformational change of NHE-1 → activation of NHE-1.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3–SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

Over 300,000 patients develop squamous cell carcinoma of the head and neck (HNSCC) worldwide with 25-30% of patients ultimately dying from their disease. Currently, molecular biomarkers are not used in HNSCC but several genes have been identified including mutant TP53 (mutp53) Our recent work has identified an approach to stratify patients with tumors harboring high or low risk TP53 mutations. Non-muscle Myosin IIA (NMIIA) was recently identified as a tumor suppressor in HNSCC. We now demonstrate that low MYH9 expression is associated with decreased survival in patients with head and neck cancer harboring low-risk mutp53 but not high-risk mutp53. Furthermore, inhibition of NMIIA leads to increased invasion in cells harboring wildtype p53 (wtp53), which was not observed in high-risk mutp53 cells. This increased invasiveness of wtp53 following NMIIA inhibition was associated with reduced p53 target gene expression and was absent in cells expressing mutp53. This reduced expression may be due, in part, to a decrease in nuclear localization of wtp53. These findings suggest that the tumor suppressor capability of wtp53 is dependent upon functional NMIIA and that the invasive phenotype of high-risk mutp53 is independent of NMIIA.

Epidermal growth factor (EGF) is linked to the pathogenesis of polycystic kidney disease (PKD). We explored signaling pathways activated by EGF in orpk cilia (-) collecting duct cell line derived from a mouse model of PKD (hypomorph of the Tg737/Ift88 gene) with severely stunted cilia, and in a control orpk cilia (+) cell line with normal cilia. RT-PCR demonstrated mRNAs for EGF receptor subunits ErbB1, ErbB2, ErbB3, ErbB4, and mRNAs for Na(+)/H(+) exchangers (NHE), NHE-1, NHE-2, NHE-3, NHE-4, and NHE-5 in both cell lines. EGF stimulated proton efflux in both cell lines. This effect was significantly attenuated by MIA, 5-(n-methyl-N-isobutyl) amiloride, a selective inhibitor of NHE-1 and NHE-2, and orpk cilia (-) cells were more sensitive to MIA than control cells (P < 0.01). EGF significantly induced extracellular signal-regulated kinase (ERK) phosphorylation in both cilia (+) and cilia (-) cells (63.3 and 123.6%, respectively), but the effect was more pronounced in orpk cilia (-) cells (P < 0.01). MIA significantly attenuated EGF-induced ERK phosphorylation only in orpk cilia (-) cells (P < 0.01). EGF increased proliferation of orpk cilia (+) cells and orpk cilia (-) cells, respectively, and MIA at 1-5 μM attenuated EGF-induced proliferation in orpk cilia (-) cells without affecting proliferation of orpk cilia (+) cells. EGF-induced proliferation of both cell lines was significantly decreased by the EGFR tyrosine kinase inhibitor AG1478 and MEK inhibitor PD98059. These results suggest that EGF exerts mitogenic effects in the orpk cilia (-) cells via activation of growth-associated amiloride-sensitive NHEs and ERK.