Volume 127, Issue 5, Pages 1423-1435 (November 2004) Regeneration of injured intestinal mucosa is impaired in hepatocyte growth factor activator-deficient mice  Hiroshi Itoh, Seiji Naganuma, Naoki Takeda, Shiro Miyata, Shunro Uchinokura, Tsuyoshi Fukushima, Shuichiro Uchiyama, Hiroyuki Tanaka, Koki Nagaike, Takeshi Shimomura, Keiji Miyazawa, Gen Yamada, Naomi Kitamura, Masashi Koono, Hiroaki Kataoka  Gastroenterology  Volume 127, Issue 5, Pages 1423-1435 (November 2004) DOI: 10.1053/j.gastro.2004.08.027 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Targeted disruption of mHGFA gene. (A) Strategy for homologous recombination. A diagram of the targeting vector (middle) was designated to replace the exon 2–4 regions of mHGFA gene in the wild-type allele (top) with a neo cassette and the 3′-flanking region with a herpes simplex thymidine kinase gene cassette. The predicted mutant allele (bottom) was generated by homologous recombination. (B) PCR analysis of tail DNA for screening. Use of PCR for both the presence of mHGFA gene (exon 1–2 region; primers P1/P3) and a neo cassette (primers P2/P4) demonstrated a single 1.5-kbp product consistent with the mutant allele, a single 0.5-kbp product with the wild-type allele, and both 1.5-kbp and 0.5-kbp products with heterozygous gene deletion. The locations and directions of the primers used are shown in A. (C) Southern blot analysis of tail DNA for reconfirmation. HindIII digestion of genomic DNA resulted in generation of a 1.5-kbp fragment unique to the homologous recombination and/or a 4.8-kbp fragment unique to the wild-type allele. A single 4.8-kbp fragment represents the wild-type mice, a single 1.5-kbp fragment represents HGFA-deficient mice, and both 4.8-kbp and 1.5-kbp fragments represent heterozygous mice. The locations of the probe used are shown in A. Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 The absence of HGFA gene expression in HGFA-deficient mice. (A) Northern blot analysis of total cellular RNA derived from the liver. No visible band corresponding to mature HGFA mRNA (approximately 2.1 kb) was detected in HGFA-deficient mice. Control glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is also shown. (B) RT-PCR analysis of the colon. No visible band corresponding to HGFA mRNA (500 base pairs; primer set P1/P5) was detected in HGFA-deficient mice. Control β-actin was also shown. M, molecular weight marker. (C) Immunoblot analysis using anti-mHGFA pAb. No detectable band corresponding to mHGFA protein (pro form; 96 kilodaltons) was found in the liver extracts of HGFA-deficient mice. Control actin was also shown. (D) HGF processing activity of the serum. Recombinant HGFA (rHGFA) was used as a positive control that shows complete processing of the inactive form of HGF (scHGF; 94 kilodaltons) to the active form of HGF (hcHGF; 62 kilodaltons). The HGF processing activity of the serum was lost in HGFA-deficient mice. Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Changes in (A) body weight and (B) survival rate with continuous administration of 3% DSS. (A) Both HGFA-deficient and wild-type mice showed progressive weight loss. (B) The survival rate was slightly low in HGFA-deficient mice, but no statistical significance was observed. Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Changes in (A) body weight and (B) survival rate with administration of 3% DSS for 6 days followed by administration of distilled water without DSS for 6 subsequent days, and (C) survival rate with rectal administration of 5% acetic acid. (A) Recovery of body weight was markedly delayed in HGFA-deficient mice compared with control wild-type mice (*P < .05 at days 9 and 10; **P < .01 at days 11 and 12). Regardless of the change to distilled water without 3% DSS on day 6, body weights were not increased in HGFA-deficient mice. (B) Twenty-eight percent (16 of 56) of HGFA-deficient mice survived on day 12, while 75% (35 of 47) of control wild-type mice survived through the same period (P < .005). (C) A total of 36.6% (15 of 41) of HGFA-deficient mice survived for 5 days after rectal administration of 5% acetic acid, while 84.2% (16 of 19) of control wild-type mice survived injury (P < .001). Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 5 (A) Histology of the colon 3 days after changing to distilled water from 3% DSS. In wild-type mice (upper 2 photos), mucosal repair by both epithelial restitution and proliferation occurs sufficiently. In HGFA-deficient mice (lower 2 photos), both epithelial restitution and proliferation were impaired and the injured mucosa was not sufficiently covered by regenerative epithelium compared with normal wild-type mice (H&E staining). (B) Impaired regeneration of injured mucosa was also observed at 2 days after rectal administration of 5% acetic acid in HGFA-deficient mice (lower 2 photos) compared with control wild-type mice (upper 2 photos) (H&E staining). Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 6 (A) Comparison of the histologic score of mucosal injury. The total activity scores of HGFA-deficient mice were significantly high (P < .001) on day 9 (DSS-induced colitis) and on day 3 (acetic acid-induced colitis) compared with those of control mice. On the other hand, the total activity scores on day 6 (DSS-induced colitis) and on day 1 (acetic acid-induced colitis) were comparable between HGFA-deficient mice and control mice (n = 10 and n = 5 for DSS-induced colitis and acetic acid-induced colitis, respectively). (B) Results of conventional RT-PCR (upper panel) and quantitative real-time RT-PCR analyses (lower panel) of HGFA and matriptase/MT-SP1 expression in the colon during the course of DSS- and acetic acid-induced colitis. HGFA mRNA was not detectable throughout the course of colitis in HGFA-deficient mice, whereas it was detected abundantly before colonic injury in control wild-type mice. The mRNA of matriptase/MT-SP1 was detected before colonic injury in both mice, and the levels were significantly decreased on day 6 (DSS-induced colitis) and on day 1 (acetic acid-induced colitis) and were slightly recovered on day 9 (DSS-induced colitis) and on day 3 (acetic acid-induced colitis). Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 7 (A) Western blot analysis for the molecular forms of HGF in the extracts derived from injured (administration of 3% DSS for 6 days) and normal colon tissues. In the injured intestinal mucosa, although the activated form of HGF (hcHGF) was observed in both HGFA-deficient and control wild-type mice, the intensity of hcHGF of control mice was significantly higher than that of HGFA-deficient mice. The inactive form of HGF (scHGF) was still observed in the injured mucosa of HGFA-deficient mice but not of control mice. The mean extent (±SE) of HGF processing of 3 independent experiments is also shown. In the injured mucosa, the mean extent of HGF processing was 79% and 35% in control mice and HGFA-deficient mice, respectively, and the difference was statistically significant (*P < 0.01 compared with control wild-type mice). (B) HGF processing activity of the extracts derived from the injured and normal colons. Eight micrograms of scHGF was mixed with colon extract and incubated for 12 hours at 37°C. Then the processing of scHGF was analyzed by Western blot as described in Materials and Methods. The mean processing activity of 3 independent experiments was also shown. HGF processing activity was very low in the extracts derived from both injured and normal colons of HGFA-deficient mice (*P < 0.01 compared with control wild-type mice). (C) HGF processing activity of the serum on colonic injury (DSS-induced colitis model on day 6) assessed by Western blot analysis as described previously. Note that control recombinant scHGF (negative control; NC) used in this study had been processed partly during the preparation. The mean of HGF processing activity in 3 independent experiments was only 1.8% (normal serum; C) and 4.1% (serum obtained from injured mice; Inj) in HGFA-deficient mice, while it was 65.7% (normal serum; C) and 91.8% (injured mice serum; Inj) in control wild-type mice (*P < 0.01). Significant processing activity was found only in the serum of control wild-type mice, which was clearly shown by the generation of light-chain of active 2-chain form HGF (2 bands observed around 32 kilodaltons due to different degrees of glycosylation). Gastroenterology 2004 127, 1423-1435DOI: (10.1053/j.gastro.2004.08.027) Copyright © 2004 American Gastroenterological Association Terms and Conditions