Volume 116, Issue 5, Pages 1081-1088 (May 1999) h-sgk serine-threonine protein kinase gene as transcriptional target of transforming growth factor β in human intestine  Siegfried Waldegger*, Karin Klingel‡, Petra Barth*, Martina Sauter‡, Martina Lanzendörfer*, Reinhard Kandolf‡, Florian Lang*  Gastroenterology  Volume 116, Issue 5, Pages 1081-1088 (May 1999) DOI: 10.1016/S0016-5085(99)70011-9 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Cellular localization of h-sgk mRNA expression in normal ileum. Radioactive in situ hybridization revealed expression of h-sgk transcripts in (A) villus enterocytes and in (B) single mononuclear cells. The arrowheads in A mark the relatively sharp transition zone between h-sgk–expressing and –nonexpressing enterocytes along the crypt-villus axis. (C) No autoradiographic signals were detected when mucosal biopsy specimens were hybridized with the α-35S–labeled RNA sense control h-sgk probe. Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 In situ detection of h-sgk mRNA in the ileum of patients with Crohn's disease. In addition to (A) apical enterocytes, (B) crypt cells and (C) mononuclear inflammatory cells revealed high levels of h-sgk transcripts. No labeling of cells was observed after hybridization with (D) sense h-sgk control RNA probe. Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 (A) Spatial correlation of h-sgk mRNA-expressing cells and immunohistochemically labeled macrophages in tissue sections of ileum revealing regional enteritis, Crohn (big arrows). Single macrophages did not express h-sgk mRNA (small arrows), and single h-sgk– positive cells showed no immunohistochemical staining (brown precipitates) for CD68 (arrowheads). (B) No unspecific labeling of immunohistochemically stained macrophages was observed after hybridization of tissue sections with sense h-sgk control RNA probe. Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Changing h-sgk transcript levels in different cell lines after stimulation with TGF-β1. (A) h-sgk transcript levels after addition of TGF-β1 (2 μg/L) to U937 macrophages for 0.5, 1, 2, 4, and 6 hours. (B) Increase of h-sgk transcript levels in U937 cells after stimulation with 1, 3, 10, and 30 μg/L TGF-β1 for 2 hours. (C) Increasing h-sgk transcript levels after addition of TGF-β1 (2 μg/L) for the indicated time periods in Intestine 407 (left) and HepG2 cells (right). Total cytoplasmic RNA (20 μg per lane) was examined for h-sgk expression by Northern blot analysis as described. Equal loading of the lanes was shown by ethidium bromide staining of the RNA in the Northern gel. An inverted picture of the 18S RNA fraction is shown. Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Increase of h-sgk expression after stimulation with TGF-β1 (+) in the absence and presence of cycloheximide. U937 cells were incubated with cycloheximide (10 mg/L) 2 hours before stimulation with TGF-β1 (2 μg/L) for an additional 2 hours in the presence of the protein synthesis inhibitor. (B) h-sgk transcript levels in U937 cells after treatment with phorbol didecanoate (P, 100 nmol/L), phorbol didecanoate and A23187 (1 μmol/L; P + A), and A23187 alone (A) for 2 hours. Lane c shows the basal expression under control conditions. Twenty micrograms of total RNA per lane was used for Northern blot analysis. The ethidium bromide–stained 18S RNA bands show equal loading of the lanes (inverted picture). Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Cellular localization of TGF-β protein expression in normal ileum. Immunohistochemical analysis revealed expression of TGF-β in villus enterocytes and in single cells of the lamina propria. (B) In the ileum of patients with Crohn's disease, in addition to apical enterocytes, crypt cells as well as mononuclear inflammatory cells revealed high levels of TGF-β expression. Gastroenterology 1999 116, 1081-1088DOI: (10.1016/S0016-5085(99)70011-9) Copyright © 1999 American Gastroenterological Association Terms and Conditions