Abstract
Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression
Highlights
Recruited to sites of integrin clustering via interactions of its C-terminal domain with integrin-associated proteins such as talin and paxillin
We confirm that Focal adhesion kinase (FAK) is required for tumor necrosis factor-␣ (TNF␣)stimulated IL-6 mRNA production and protein secretion by comparisons of FAKϪ/Ϫ and FAKϩ/ϩ fibroblasts as was first shown by Funakoshi-Tago et al (14)
We found that all aspects of TNF␣ induced nuclear factor-B (NF-B) activation, including IB␣ degradation, NF-B nuclear translocation, NF-B DNA binding, and stimulated NF-B transactivation activity, are equivalent in FAKϩ/ϩ, FAKϪ/Ϫ, FAKϩ/ϩ, FAK short hairpin RNA (shRNA), and FAKϪ/Ϫ Pyk2 shRNA-expressing fibroblasts
Summary
Cell Culture—FAKϩ/ϩ, FAKϪ/Ϫ, FAK-reconstituted (clone DP3), SYF (SrcϪ/Ϫ, c-YesϪ/Ϫ, FynϪ/Ϫ), and SYF ϩ c-Src murine embryonic fibroblasts were used as described (23–25). 4T1 breast carcinoma cells, A549 lung adenocarcinoma cells, and PC-3 prostate carcinoma cells were from ATCC (Manassas, VA). Cell Culture—FAKϩ/ϩ, FAKϪ/Ϫ, FAK-reconstituted (clone DP3), SYF (SrcϪ/Ϫ, c-YesϪ/Ϫ, FynϪ/Ϫ), and SYF ϩ c-Src murine embryonic fibroblasts were used as described (23–25). 4T1 FAK shRNA and scrambled shRNA control cells were generated as described (27). Viral Expression—The pLentiLox shRNA vector was used to create stable knockdown of FAK expression in NB8, PC3, and A549 carcinoma cells as described (20). For murine Pyk shRNA, the oligonucleotides forward 5Ј-tgaagtagttcttaaccgcattcaagagatgcggttaagaactacttcttttttc-3Ј and reverse 5Ј-tcgagaaaaaagaagtagttcttaaccgcatctcttgaatgcggttaagaactacttca-3Ј were cloned into pLentiLox. Cells were sorted for GFP expression, and the shRNA efficacy was verified by immunoblotting. FAKϪ/Ϫ fibroblasts and A549 carcinoma cells were selected in puromycin (5 g/ml), and CA-MEK1 expression was verified by immunoblotting. Fluorescence-activated Cell Sorting Analysis—FAKϩ/ϩ and FAKϪ/Ϫ fibroblasts were trypsinized, fixed with 3.7% formaldehyde solution for 10 min at room temperature, and incubated with phycoerythrin-conjugated anti-tumor necrosis factor receptor-1 (TNFR1) or TNFR2 antibodies.
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