Glucagon-induced expression of the MAP kinase phosphatase MKP-1 in rat hepatocytes  Freimut Schliess, Anna Kordelia Kurz, Dieter Häussinger  Gastroenterology  Volume 118, Issue 5, Pages 929-936 (May 2000) DOI: 10.1016/S0016-5085(00)70179-X Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 Induction of MKP-1 mRNA expression by glucagon or 8-CPT-cAMP. Hepatocytes were exposed to glucagon (G, 100, 50, or 10 nmol/L) or 8-CPT-cAMP (cA, 50 or 10 μmol/L) or remained untreated (Control) for the time period indicated. The cells were lysed, and total RNA was analyzed by Northern blotting using the cDNA probe for MKP-1. mRNA levels were normalized to GAPDH mRNA levels; the values found in the respective control condition were set to 1. A representative of 3 independent experiments. Gastroenterology 2000 118, 929-936DOI: (10.1016/S0016-5085(00)70179-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Induction of MKP-1 protein expression by glucagon or 8-CPT-cAMP. Hepatocytes were exposed to glucagon (G, 100 nmol/L) or 8-CPT-cAMP (cA, 50 μmol/L) or remained untreated for the time period indicated. For control, H4IIE hepatoma cells were exposed to 43°C for 1 hour and were allowed to recover at 37°C for 1 hour. Alternatively, the heat shock was substituted by 1-hour incubation of 37°C. After treatment, cells were lysed and the protein extract was analyzed by Western blotting using an antibody recognizing MKP-1. A representative of 3 independent experiments. Gastroenterology 2000 118, 929-936DOI: (10.1016/S0016-5085(00)70179-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 Characterization of the glucagon-induced pathway to MKP-1 mRNA induction. Total mRNA of treated or untreated (control) hepatocytes was analyzed for the presence of MKP-1 mRNA in Northern blot analysis using the cDNA probe for MKP-1. mRNA levels were normalized to GAPDH mRNA levels; the values found in the respective control condition (control) were set to 1. Information on significances is given in the text. Representatives of 3 independent experiments. (A) Induction of MKP-1 mRNA expression by agents increasing intracellular cAMP concentration. Hepatocytes remained untreated (Control) or were incubated with Sp-5,6-DCI-cBiMPS (50 μmol/L), isoproterenol (10 μmol/L), or forskolin (50 μmol/L) plus isobutylmethylxanthine (IBMX, 100 μmol/L) for 30 minutes. (B) Role of cyclic nucleotide–dependent kinases for glucagon-induced MKP-1 mRNA expression. Hepatocytes remained untreated (controls, lanes 1 and 7 ) or were exposed to glucagon (G, 100 nmol/L, lanes 2 and 8) or dibutyryl-cGMP (dB-cGMP, 0.5 mmol/L, lane 10) for 30 minutes without further treatment or after a 30-minute preincubation period with H7 (10 μmol/L, lane 3), SQ22536 (0.5 mmol/L, lane 4), H89 (1 μmol/L, lane 5), KT5720 (1 μmol/L, lane 6), and Rp-8-CPT-cGMPS (100 μmol/L, lanes 9 and 11). (C) Role of MAP kinases for glucagon-induced MKP-1 expression. Hepatocytes remained untreated (lane1) or were exposed to glucagon (G, 100 nmol/L) without further treatment (lane 2) or after a 30-minute preincubation with PD098059 (10 μmol/L, lane 3) and SB203580 (20 μmol/L, lane 4). (D) Role of Ca2+ for glucagon-induced MKP-1 mRNA expression. Hepatocytes remained untreated (control, lanes 1 and 6) or were exposed to glucagon (G, 100 nmol/L, lanes 2 and 7 ), PMA (1 μmol/L, lane 8), ionomycin (1 μmol/L, lane 10 ), or okadaic acid (OA, 100 nmol/L, lane 11) for 30 minutes without further pretreatment or after a 30-minute preincubation period with BAPTA-AM (10 μmol/L, lane 3), EGTA (3 mmol/L, lane 4), and Gö6850 (1 μmol/L, lanes 5 and 9). (E ) Role of cell shrinkage, oxidative stress, or tyrosine phosphatases for glucagon-induced MKP-1 mRNA expression. Hepatocytes remained untreated (control, lane 1) or were incubated with glucagon (G, 100 nmol/L) for 30 minutes without further treatment (lane 2), with a simultaneous reduction of osmolarity from 305 mOsm/L (normosmolarity) to hyposmolarity (205 mOsm/L, lane 3) or after a 30-minute preincubation period with trolox (50 μmol/L, lane 4), BHT (10 μmol/L, lane 5 ), or sodium orthovanadate (V, 100 μmol/L, lane 6). Cells were also exposed to hyposmolarity (205 mOsm/L) for 30 minutes (lane 7 ) or to vanadate (V, 100 μmol/L, lane 8) for 1 hour. Hyposmolarity was achieved by dilution of the medium with the appropriate volume of NaCl-free medium. Gastroenterology 2000 118, 929-936DOI: (10.1016/S0016-5085(00)70179-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Cross talk between glucagon- and EGF-induced signaling. Hepatocytes were stimulated without pretreatment with 10 ng/mL EGF for 10 minutes (lanes 2, 6, and 10). Alternatively, glucagon (G, 100 nmol/L) was present 5, 15, or 60 minutes before and during EGF treatment as indicated (lanes 3, 7, and 11), resulting in a glucagon exposure for 15, 25, or 70 minutes as indicated. Control cells remained untreated (lanes 1, 5, and 9) or were exposed only to glucagon (lanes 4, 8, and 12). After the end of incubations, cells were harvested and protein extracts were used for measuring Erk-1/Erk-2 phosphorylation by Western blot analysis and Erk-1/Erk-2 activation with the immune complex assay, using an antibody raised against Erk-1 and Erk-2 and myelin basic protein (MBP) as substrate. Representatives of 3 independent experiments. Gastroenterology 2000 118, 929-936DOI: (10.1016/S0016-5085(00)70179-X) Copyright © 2000 American Gastroenterological Association Terms and Conditions