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The MUC2 gene 5′-flanking sequence directs goblet cell-specific expression in the small intestine (SI) of transgenic mice
Intestinal Disorders A375
April 1998 fragments (p94 and p84) after 45 and 120 minutes, respectively, as demonstrated by Western blot. 0
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120:180
240 (min)
:!~!i,~:~ii,~:~.!~:~i-~i i: ::!? ~,-~ !! !i~i!~! ~::!ii i~:!:i'!:!!~:i~i :~'i I!!~!~,,,:~ ::%:!! : ~!!:ii!
- p 94
!. p 84
FAK cleavage occurred simultaneously with the activation of caspase 3. Inhibition of caspase 3 family members by addition of DEVD-CHO (1.5 [aM) completely blocked FAK cleavage, 100-fold more effectively than inhibition of caspase 1 family members with YVAD-CHO (150 jaM). In contrast, RAFTK, another component of the integrin-linked signaling complex, is expressed in these cells but remains uncleaved during anoikis. These results demonstrate that FAK, but not RAFTK, is selectively cleaved into distinct fragments by a proteolytic process involving caspase 3 family members. The rapid proteolysis of FAK after detachment of IEC may disrupt the architecture and function of focal adhesions and thereby provide an effective mechanism to prevent re-attachment and ectoptic growth of IEC in vivo after these cells exfoliate. • G1530 LOSS OF ANCHORAGE LEADS TO EXTENSIVE APOPTOSIS OF HUMAN INTESTINAL EPITHELIAL CELLS (IEC), INDEPENDENT O F THEIR LOCATION ON THE CRYPT-VILLUS AXIS OR THE MODE OF ISOLATION. J. Grossmann. G. Latella*, C. Fiocchi, A.D. Levine. Case Western Reserve University School of Medicine, Cleveland, Ohio and *University of L'Aquila, Italy. Loss of anchorage to the substratum induces a form of apoptosis, termed detachment-induced cell death or 'anoikis.' In vivo IEC die of apoptosis as they reach the lumen and are shed, a process which may resemble anoikis. In vitro IEC die of apoptosis after detachment during isolation, making longterm primary culture of human IEC a goal not reached. In this study we evaluate the susceptibility of different IEC compartments to anoikis and the contribution of the isolation technique to the extent of apoptosis. IEC were isolated from colonic surgical specimens by different, previously described methods (protocol A and B: chelating agents EDTA and EGTA, protocol C: dispase and protocol D: mechanical disruption). IEC from different compartments (LU=lumen, MI=mid axis, CB=crypt base) of the crypt-villus axis were isolated by sequential dispase washes as described. Freshly isolated IEC were kept in suspension and evaluated by acridine orange/ethidium bromide stain for populations of viable, necrotic and apoptotic ceils three hours after isolation. Fragmentation of DNA was assessed by gel electrophoresis. Less than 3% of IEC were viable by acridine orange/ethidium bromide stain after three hours of suspension, regardless of the isolation protocol employed. 10-25% of IEC were necrotic and all remaining IEC (75-95%) displayed unmistakable evidence of apoptosis since nuclear beads, containing condensed DNA, are clearly identified with this stain. The prevalence of apoptosis was confirmed by DNA gel electrophoresis. protocol necrotic (%) apoptotic (%) viable (%)
A 12 87 1
B 13 85 2
C 15 82 3
D 25 84 1
IEC origin LU necrotic(%) 14 apoptotic(%) 85 viable (%) 1
MI 10 87 3
CB 12 85 3
No difference in the degree of apoptosis was observed in cells isolated from the lumen, mid axis or crypt base. Greater than 85% of IEC had died of apoptosis and less than 5% were viable. This observation was confirmed by DNA gel electrophoresis. These results demonstrate that the vast majority of IEC will rapidly die of apoptosis after loss of anchorage. Minimizing the time of detachment during isolation and preserving cell-cell attachment within the epithelium may be the avenue to successful primary culture of non-transformed human IEC. Even though IEC at the base of the crypt express higher levels of bcl-2, detachment leading to apoptosis overcomes the intrinsic cytoprotection of these cells.
• G1531 CHOLYLSARCOSINE (CS) IMPROVES FAT ABSORPTION IN SHORT BOWEL SYNDROME (SBS):. Christine Gruy-Kapral, Katherine H Little, John S Fordtran, Lee R Hagey, and Alan F Hofmann, Baylor University Medical Center, Dallas, "IX and University of California, San Diego, CA Current therapy for SBS other than TPN includes secretion inhibitors (PPI, octreotide), diet alteration, and epithelial growth factors (growth hormone, glutamine). Bile acid replacement therapy is not used, even though fat malabsorption in SBS is due in part to impaired micellar solubilization because of bile acid malabsorption. No conjugated bile acid product is presently available on the US market. CS, a conjugated bile acid analogue, is resistant to deconjugation and dehydroxylation, and has no secretory properties. We tested whether CS in capsules, ingested with meals, would improve fat absorption in a SBS patient (resected ileum and colon) who was extremely malnourished and in whom TPN could not be used because of a hypercoagulable condition. A metabolic balance study was conducted. Fat intake was 124 g/d and study periods were 3 days. Ileostomy output was analyzed daily. Treatment
Control period CS (6 g/d) Control period CS (12 g/d)
Fecal Fat g/d 70 34 55 26
Fat Absorption g/d 54 90 69 98
Fecal N g/d 6.6 5.2 5. l 5.2
Fecal Calcium mEq/d 23 13 24 20
Fecal Weight g/d 2115 2009 1784 2029
CS improved fat absorption (p=0.002) without increasing the volume of ileostomy output. Caloric absorption from fat increased by about 300 kCal/d. Fecal calcium output fell (p=0.007), indicating increased calcium absorption. As expected, there was no effect of CS on nitrogen output. Conclusion: CS improved fat and calcium absorption in a patient with SBS with ileostomy. In our opinion, conjugated bile acid replacement therapy should be considered in the nutritional management of SBS. Dr. Hofmann previously had a patent on CS, but it has expired. G1532 THE MUC2 GENE 5'-FLANKING SEQUENCE DIRECTS GOBLET CELL-SPECIFIC EXPRESSION IN THE SMALL INTESTINE (SI) OF TRANSGENIC MICE. James R. Gum, Jr., James W. Hicks, Satyajit Karnik, Joe C. Hong, and Young S. Kim. GI Research Lab, VA Med. Center & Depts. of Anat. and Med., UCSF, San Francisco, CA. While several detailed studies have yielded considerable insight into the mechanisms regulating gene expression in intestinal absorptive cells, there is a sparsity of such information pertaining to the other cell lineages of the intestinal epithelium. In the present study, we examined the expression of a major goblet cell mucin gene (MUC2) using transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Methods: Standard techniques were used to produce and analyze mice containing the -2864 to +17 MUC2-hGH promoter/reporter transgene. DNA and RNA blot analysis and RNase protection assays were performed as previously in our laboratory. Immunogold silver staining was used to localize transgene expression in histological sections with goblet cells being localized by Alcian blue counterstaining. Results: Eight founder mice were obtained containing between 1 and 32 copies of the transgene. Reporter mRNA expression levels in the different pedigrees were highly variable and not strictly dependent upon copy number, however the pattern of expression was similar, with middle to distal SI expressing high levels of reporter and low to undetectable expression occurring in the colon. In contrast, native MUC2 expression was highest in the colon. Almost no hGH reporter mRNA or native MUC2 mRNA expression was detectable in non-GI tract tissues other than in the proximal tracheal glands. RNase protection analysis indicated a similar transcription start site for the transgene as for native MUC2. Immunohistochemical analysis indicated goblet cell specific expression of transgene beginning in the lower portions of the crypts and extending to the lower portion of the villi while in situ hybridization analysis indicates that goblet ceils throughout the crypts and villi express MUC2 message. Conclusions: 1) The -2864 to +17 region of the MUC2 gene 5'-flanking sequence is sufficient for gobletcell specific expression of MUC2 in the SI. 2) Elements outside of this region are required for high level colonic expression. 3) Reporter expression begins with deep crypt goblet cells but does not extend to the upper reaches of the villi, suggesting the transgene lacks an element required for sustaining expression of MUC2 during goblet cell migration to the villi tips. 4) The marked integration site dependence for transgene expression between pedigrees suggests a possible regulatory significance for the clustering of secretory mucin genes found at chromosome 1 lp15. This work supported by the VA Medical Research Service, the Betz Foundation and NCI Grant CA 24321.
April 1998 fragments (p94 and p84) after 45 and 120 minutes, respectively, as demonstrated by Western blot. 0
15
30
45
60
90
120:180
240 (min)
:!~!i,~:~ii,~:~.!~:~i-~i i: ::!? ~,-~ !! !i~i!~! ~::!ii i~:!:i'!:!!~:i~i :~'i I!!~!~,,,:~ ::%:!! : ~!!:ii!
- p 94
!. p 84
FAK cleavage occurred simultaneously with the activation of caspase 3. Inhibition of caspase 3 family members by addition of DEVD-CHO (1.5 [aM) completely blocked FAK cleavage, 100-fold more effectively than inhibition of caspase 1 family members with YVAD-CHO (150 jaM). In contrast, RAFTK, another component of the integrin-linked signaling complex, is expressed in these cells but remains uncleaved during anoikis. These results demonstrate that FAK, but not RAFTK, is selectively cleaved into distinct fragments by a proteolytic process involving caspase 3 family members. The rapid proteolysis of FAK after detachment of IEC may disrupt the architecture and function of focal adhesions and thereby provide an effective mechanism to prevent re-attachment and ectoptic growth of IEC in vivo after these cells exfoliate. • G1530 LOSS OF ANCHORAGE LEADS TO EXTENSIVE APOPTOSIS OF HUMAN INTESTINAL EPITHELIAL CELLS (IEC), INDEPENDENT O F THEIR LOCATION ON THE CRYPT-VILLUS AXIS OR THE MODE OF ISOLATION. J. Grossmann. G. Latella*, C. Fiocchi, A.D. Levine. Case Western Reserve University School of Medicine, Cleveland, Ohio and *University of L'Aquila, Italy. Loss of anchorage to the substratum induces a form of apoptosis, termed detachment-induced cell death or 'anoikis.' In vivo IEC die of apoptosis as they reach the lumen and are shed, a process which may resemble anoikis. In vitro IEC die of apoptosis after detachment during isolation, making longterm primary culture of human IEC a goal not reached. In this study we evaluate the susceptibility of different IEC compartments to anoikis and the contribution of the isolation technique to the extent of apoptosis. IEC were isolated from colonic surgical specimens by different, previously described methods (protocol A and B: chelating agents EDTA and EGTA, protocol C: dispase and protocol D: mechanical disruption). IEC from different compartments (LU=lumen, MI=mid axis, CB=crypt base) of the crypt-villus axis were isolated by sequential dispase washes as described. Freshly isolated IEC were kept in suspension and evaluated by acridine orange/ethidium bromide stain for populations of viable, necrotic and apoptotic ceils three hours after isolation. Fragmentation of DNA was assessed by gel electrophoresis. Less than 3% of IEC were viable by acridine orange/ethidium bromide stain after three hours of suspension, regardless of the isolation protocol employed. 10-25% of IEC were necrotic and all remaining IEC (75-95%) displayed unmistakable evidence of apoptosis since nuclear beads, containing condensed DNA, are clearly identified with this stain. The prevalence of apoptosis was confirmed by DNA gel electrophoresis. protocol necrotic (%) apoptotic (%) viable (%)
A 12 87 1
B 13 85 2
C 15 82 3
D 25 84 1
IEC origin LU necrotic(%) 14 apoptotic(%) 85 viable (%) 1
MI 10 87 3
CB 12 85 3
No difference in the degree of apoptosis was observed in cells isolated from the lumen, mid axis or crypt base. Greater than 85% of IEC had died of apoptosis and less than 5% were viable. This observation was confirmed by DNA gel electrophoresis. These results demonstrate that the vast majority of IEC will rapidly die of apoptosis after loss of anchorage. Minimizing the time of detachment during isolation and preserving cell-cell attachment within the epithelium may be the avenue to successful primary culture of non-transformed human IEC. Even though IEC at the base of the crypt express higher levels of bcl-2, detachment leading to apoptosis overcomes the intrinsic cytoprotection of these cells.
• G1531 CHOLYLSARCOSINE (CS) IMPROVES FAT ABSORPTION IN SHORT BOWEL SYNDROME (SBS):. Christine Gruy-Kapral, Katherine H Little, John S Fordtran, Lee R Hagey, and Alan F Hofmann, Baylor University Medical Center, Dallas, "IX and University of California, San Diego, CA Current therapy for SBS other than TPN includes secretion inhibitors (PPI, octreotide), diet alteration, and epithelial growth factors (growth hormone, glutamine). Bile acid replacement therapy is not used, even though fat malabsorption in SBS is due in part to impaired micellar solubilization because of bile acid malabsorption. No conjugated bile acid product is presently available on the US market. CS, a conjugated bile acid analogue, is resistant to deconjugation and dehydroxylation, and has no secretory properties. We tested whether CS in capsules, ingested with meals, would improve fat absorption in a SBS patient (resected ileum and colon) who was extremely malnourished and in whom TPN could not be used because of a hypercoagulable condition. A metabolic balance study was conducted. Fat intake was 124 g/d and study periods were 3 days. Ileostomy output was analyzed daily. Treatment
Control period CS (6 g/d) Control period CS (12 g/d)
Fecal Fat g/d 70 34 55 26
Fat Absorption g/d 54 90 69 98
Fecal N g/d 6.6 5.2 5. l 5.2
Fecal Calcium mEq/d 23 13 24 20
Fecal Weight g/d 2115 2009 1784 2029
CS improved fat absorption (p=0.002) without increasing the volume of ileostomy output. Caloric absorption from fat increased by about 300 kCal/d. Fecal calcium output fell (p=0.007), indicating increased calcium absorption. As expected, there was no effect of CS on nitrogen output. Conclusion: CS improved fat and calcium absorption in a patient with SBS with ileostomy. In our opinion, conjugated bile acid replacement therapy should be considered in the nutritional management of SBS. Dr. Hofmann previously had a patent on CS, but it has expired. G1532 THE MUC2 GENE 5'-FLANKING SEQUENCE DIRECTS GOBLET CELL-SPECIFIC EXPRESSION IN THE SMALL INTESTINE (SI) OF TRANSGENIC MICE. James R. Gum, Jr., James W. Hicks, Satyajit Karnik, Joe C. Hong, and Young S. Kim. GI Research Lab, VA Med. Center & Depts. of Anat. and Med., UCSF, San Francisco, CA. While several detailed studies have yielded considerable insight into the mechanisms regulating gene expression in intestinal absorptive cells, there is a sparsity of such information pertaining to the other cell lineages of the intestinal epithelium. In the present study, we examined the expression of a major goblet cell mucin gene (MUC2) using transgenic mice containing bases -2864 to +17 of the human MUC2 5'-flanking region fused into the 5'-untranslated region of a human growth hormone (hGH) reporter gene. Methods: Standard techniques were used to produce and analyze mice containing the -2864 to +17 MUC2-hGH promoter/reporter transgene. DNA and RNA blot analysis and RNase protection assays were performed as previously in our laboratory. Immunogold silver staining was used to localize transgene expression in histological sections with goblet cells being localized by Alcian blue counterstaining. Results: Eight founder mice were obtained containing between 1 and 32 copies of the transgene. Reporter mRNA expression levels in the different pedigrees were highly variable and not strictly dependent upon copy number, however the pattern of expression was similar, with middle to distal SI expressing high levels of reporter and low to undetectable expression occurring in the colon. In contrast, native MUC2 expression was highest in the colon. Almost no hGH reporter mRNA or native MUC2 mRNA expression was detectable in non-GI tract tissues other than in the proximal tracheal glands. RNase protection analysis indicated a similar transcription start site for the transgene as for native MUC2. Immunohistochemical analysis indicated goblet cell specific expression of transgene beginning in the lower portions of the crypts and extending to the lower portion of the villi while in situ hybridization analysis indicates that goblet ceils throughout the crypts and villi express MUC2 message. Conclusions: 1) The -2864 to +17 region of the MUC2 gene 5'-flanking sequence is sufficient for gobletcell specific expression of MUC2 in the SI. 2) Elements outside of this region are required for high level colonic expression. 3) Reporter expression begins with deep crypt goblet cells but does not extend to the upper reaches of the villi, suggesting the transgene lacks an element required for sustaining expression of MUC2 during goblet cell migration to the villi tips. 4) The marked integration site dependence for transgene expression between pedigrees suggests a possible regulatory significance for the clustering of secretory mucin genes found at chromosome 1 lp15. This work supported by the VA Medical Research Service, the Betz Foundation and NCI Grant CA 24321.