Volume 128, Issue 5, Pages 1278-1291 (May 2005) Tumor Necrosis Factor α Blockade Restores Growth Hormone Signaling in Murine Colitis  Lisa M. Difedele, Jiman He, Erin L. Bonkowski, Xiaonan Han, Matthew A. Held, Alan Bohan, Ram K. Menon, Lee A. Denson  Gastroenterology  Volume 128, Issue 5, Pages 1278-1291 (May 2005) DOI: 10.1053/j.gastro.2005.02.003 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Serum IGF-1 and liver IGF-1, ALS, and IGFBP-3 expression in colitis. (A) Serum IGF-1 was determined in WT (+/+) and IL-10 null (−/−) mice and is shown. Liver RNA was isolated from WT (+/+) and IL-10 null (−/−) mice and the (B) IGF-1 expression for the total, exon 1, and exon 2 transcripts, (C) ALS expression, and (D) IGFBP-3 expression were determined by real-time PCR. Data are shown as the mean ± SD, n = 8. Significance was tested by 1-way ANOVA and independent samples t test. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 GH-dependent hepatic STAT5 phosphorylation and DNA binding. Nuclear protein was isolated from liver of WT (+/+) and IL-10 null (−/−) mice 30 minutes after PBS or rat GH administration (1 mcg/g, IP). (A) Abundance of tyrosine phosphorylated STAT5 (ptyrSTAT5) was determined by immunoblot; shptp1 was used to confirm equal loading. (B) STAT5b DNA binding was determined by EMSA. (C) Specific competition (comp) with an excess of the unlabelled STAT5 oligonucleotide and supershift using STAT5b or Sp3 antibodies (ab) for the GH-induced STAT5 complex (gh) are shown. The specific shifted complex is identified with the STAT5 label and arrow, which also identifies the location of the depleted complex after incubation with the STAT5b antibody. Signal intensity was determined by densitometry. Data are shown as the mean ± SD, n = 6. Significance was tested by independent samples t test. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Liver GHR expression in colitis. Liver protein and RNA were isolated from WT (+/+) and IL-10 null (−/−) mice and (A) GHR protein or (B) total GHR and L2 transcript RNA expression were determined by immunoblot or real-time PCR, respectively. (A) Signal intensity was determined by densitometry; actin was used to confirm equal loading. Data are shown as the mean ± SD, (A) n = 6 and (B) n = 16. Significance was tested by independent samples t test. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Liver Sp3 nuclear abundance and DNA binding in colitis. Liver nuclear protein was isolated from WT (+/+) and IL-10 null (−/−) mice and (A) Sp3 nuclear abundance or (B) DNA binding to the GHR L2 transcript L2A cis element was determined by immunoblot or EMSA, respectively. Shptp1 was used to confirm equal loading. Signal intensity was determined by densitometry and data are shown as the mean ± SD, n = 6. Significance was tested by independent samples t test. (C) Competition (comp) with an excess of unlabelled L2A or STAT1 oligonucleotides and supershift using Sp1, Sp3, or STAT1 antibodies (antibody) for the L2A complex is shown. (B, C) The specific shifted complex is identified with the L2A label and arrow, which also identifies the location of the depleted complex after incubation with the Sp3 antibody in C. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 TNFα and IL-6 regulation of GH signaling in hepatocytes. H4IIE rat hepatoma cells were treated with rat GH (500 ng/mL for 30 min), TNFα or IL-6 (100 ng/mL for 16 h), or pretreated with TNFα or IL-6 for 16 hours followed by rat GH for 30 minutes. (A, C) Nuclear abundance of tyrosine phosphorylated STAT5 (ptyrSTAT5) was determined by immunoblot; shptp1 was used to confirm equal loading. (B, D) GHR protein abundance was determined by immunoblot; actin was used to confirm equal loading. Signal intensity was determined by densitometry. Data are shown as the mean ± SD, (A, B) n = 5 or (C, D) 3. Significance was tested by independent samples t test. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 TNFα blockade up-regulates liver GHR abundance and restores GH activation of STAT5. IL-10 null mice and WT controls were treated with PBS, with isotype control IgG antibody (1 mg, IP) weekly for 3 weeks (IgG3W), with anti-TNFα antibody (1 mg, IP) for 24 hours (aTNF24H), or weekly for 3 weeks (aTNF3W). (B, C) Mice also were treated with GH, 1 mcg/g IP 30 minutes before death. Liver protein was isolated and (A) GHR or (B) tyrosine phosphorylated STAT5 (pSTAT5) abundance were determined by immunoblot, with actin or shptp1 used to confirm equal loading, respectively. (C) STAT5b DNA binding was determined by EMSA using nuclear protein isolated from IL-10 null mice treated with isotype control antibody (IgG3W) or anti-TNFα antibody (aTNF) and WT controls treated with isotype control antibody (WT). Signal intensity was determined by densitometry and is shown as the mean ± SD, n = 4. (D) Serum IGF-1 was determined in WT (+/+) and IL-10 null (−/−) mice after PBS or anti-TNFα antibody administration, as shown. Significance was tested by 1-way ANOVA and independent samples t test, n = 4. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Effect of TNFα blockade on colon histology. IL-10 null mice were treated with PBS, isotype control antibody (IgG3W), or anti-TNFα antibody (1 mg, IP) for 24 hours (aTNF24H) or weekly for 3 weeks (aTNF3W). Colon histology scores were determined and are shown as the mean ± SD, n = 4. Significance was tested by 1-way ANOVA and independent samples t test. Gastroenterology 2005 128, 1278-1291DOI: (10.1053/j.gastro.2005.02.003) Copyright © 2005 American Gastroenterological Association Terms and Conditions