Figure 7.

Autophagy is required for TNF mediated CCL2 transcription through MAPK1/3-AP1 signaling, and the MAPK1/3 activity is controlled by the AMPK-BRAF axis. (A) Schematic representation of the (dis)AP1, NFKB, and (pro)AP1/SP1 sites of the human CCL2 promoter. Red nucleotides indicate mutations made in the human CCL2 promoter. (B) Reporter assay for the Human CCL2 promoter with wild-type sequence transfected into HaCaT cells with shCON or shATG5, following treatment with or without TNF for 24 h. (C) Same as B except that HaCaT cells were transfected with the human CCL2 promoter with wild-type sequence or mutation of the (dis)AP1 site, NFKB site, or (pro)AP1/SP1 site. (D) Same as B except that HaCaT cells were transfected with the human CCL2 promoter with mutation of NFKB site, following treatment with or without TNF for 24 h. (E) Same as D except that HaCaT cells were transfected with the human CCL2 promoter with mutation of (pro)AP1/SP1 site. (F) Same as B except that HaCaT cells were transfected with promoters with a repeated sequence of the NFKB-RE (NFKB response element) or AP1-RE (AP1 response element). (G) Same as B except that the cells were treated with PD98059 (20 µM) 2 h prior to and during TNF treatment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; compared with its corresponding Veh group. ^#, P < 0.05; ^##, P < 0.01; compared with shCON group (B, D-G), or with WT/TNF group (C); Student’s t-test. (H and I) Immunoblot analysis of p-MAPK1/3, MAPK1/3, ATG12–ATG5, and ATG7 in HaCaT cells infected with shCON, shATG5 (H), or shATG7 (I), starved overnight, followed by treatment with or without TNF (100 ng/ml) over a time course. (J and K) Immunoblot analysis of p-MAPK1/3, ATG12–ATG5, and ATG7 in NHEK cells transfected with siNC, siATG5 (J), or siATG7(K), starved with 5 fold-diluted the complete medium for NHEK for 24 h, treated with or without TNF (100 ng/ml) for 30 min. (L) Immunoblot analysis of p-BRAF (S729), BRAF, p-PRKAA1, PRKAA1, p-CRTC1, CRTC1, and MAP1LC3B/LC3B-I/II (microtubule-associated protein 1 light chain 3 beta) in HaCaT cells infected with shCON, shATG5, or shATG7. (M) Immunoblot analysis for p-BRAF, BRAF, p-MAPK1/3, and MAPK1/3 in HaCaT cells infected with shCON, shATG5, or shATG7, starved overnight, and treated with the AMPK inhibitor compound C (AMPKi 1 μM) for 2 h, followed by treatment with TNF (100 ng/ml) for 15 min. (N) Real-time PCR analysis for CCL2 mRNA level in HaCaT cells treated with or without Compound C (1 μM) for 5 h and then treated with TNF (100 ng/ml) for 24 h. Data are shown as mean ± S.E. (n ≥ 3). *P < 0.05; **P < 0.01; compared with the corresponding group without compound C treatment; Student’s t-test (C). (O) Immunoblot analysis of p-PRKAA1, PRKAA1, p-MAPK1/3, MAPK1/3, and GAPDH in HaCaT cells treated with or without TNF (100 ng/ml) or CCL2 (100 ng/ml) for 15 min. Results were obtained from at least three independent experiments