Abstract
In this study, we present multiple lines of evidence to support a critical role for heparin-bound EGF (epidermal growth factor)-like growth factor (HB-EGF) and tumor necrosis factor-alpha-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT(2A) receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells. ERK phosphorylation was attenuated by 1) neutralizing EGFR antibodies and the EGFR kinase inhibitor, AG1478, 2) neutralizing HB-EGF, but not amphiregulin, antibodies, heparin, or CM197, and 3) pharmacological inhibitors of matrix-degrading metalloproteinases or TACE small interfering RNA. Exogenously administered HB-EGF stimulated ERK phosphorylation. Additionally, TACE was co-immunoprecipitated with HB-EGF. Small interfering RNA against TACE also blocked 5-HT-induced increases in ERK phosphorylation, HB-EGF shedding, and DNA content. In aggregate, this work supports a pathway map that can be depicted as follows: 5-HT --> 5-HT(2A) receptor --> TACE --> HB-EGF shedding --> EGFR --> ERK --> increased DNA content. To our knowledge, this is the first time that TACE has been implicated in 5-HT-induced EGFR transactivation or in proliferation induced by a G protein-coupled receptor in native cells in culture.
Highlights
We present multiple lines of evidence to support a critical role for heparin-bound EGFlike growth factor (HB-epidermal growth factor (EGF)) and tumor necrosis factor-␣-converting enzyme (TACE) (ADAM17) in the transactivation of EGF receptor (EGFR), ERK phosphorylation, and cellular proliferation induced by the 5-HT2A receptor in renal mesangial cells. 5-hydroxy-tryptamine (5-HT) resulted in rapid activation of TACE, HB-EGF shedding, EGFR activation, ERK phosphorylation, and longer term increases in DNA content in mesangial cells
We present multiple lines of evidence that support a central role for TACE (ADAM17) and HB-EGF in the transactivation of EGF receptors by the 5-HT2A receptor in renal mesangial cells and in cellular proliferation
Stimulation of the 5-HT2A receptor leads to rapid activation of TACE, shedding of heparin-bound EGF-like growth factor (HBEGF), and activation of EGFRs
Summary
Chemicals and Antibodies—All cell culture media, fetal bovine serum and antibiotics, 4 –12% acrylamide gels, SDS sample buffer, and sample reducing agent were purchased from Invitrogen. One small hairpin RNA construct that resulted in nearly complete inhibition of TACE expression in human mesangial cells was chosen for use in our studies. Cell Signaling Experiments—For ERK phosphorylation studies, human and rat mesangial cells were seeded into 12-well culture plates (Falcon) and serum-starved for 2 days in 0.5% bovine serum albumin-containing medium. Resolved proteins were transferred to 0.45-m polyvinylidene difluoride membranes (Millipore), blocked with 4% nonfat dry milk or 3% bovine serum albumin for 1 h, and incubated overnight at 4 °C with the following antibodies: 1:1000 ERK and phospho-ERK (Cell Signaling), 1 g/ml HB-EGF (Oncogen), 1:500 ADAM9 (Zymed Laboratories Inc.) and ADAM10 (Chemicon), 1:1000 EGFR (Cell Signaling), 1:3000 ␣-actinin (Santa Cruz Biotechnology), or 1:2000 TACE (R&D Systems) antibodies. Statistical Analysis—Student’s t test and analysis of variance using GraphPad statistics software were performed to determine statistical significance. p values Ͻ 0.05 were regarded as statistically significant
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