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PACAP-induced growth of rat pancreatic acinar tumor cells involves increased expression of CFOS and CJUN and activation of the AP1 transcription factor-complex
April
1995
• PUTRESCINE STIMULATES TRANSCRIPTION OF mRNAs CONTAINING THE BC-1 S E Q U E N C E . . J - L Scemama*. V. Rajah. P Brachet and ER Seidel. Depts of Physiology and Biology*, East Carolina University School of Medicine, Greenville NC 27858. The potyamine putrescine is imimately involved in regulation of mucosal growth, however the molecular mechanism by which it exerts control over proliferation remains unknown. We have employed the differential display assay (DDA) to examine polyamine-regulated gene transcription. IEC-6 cells were plated in DMEM containing 5 mM difluoromethylornithine (DFMO). After 72 hr, 10 ktM pntrescine was added to one half of the culture plates and 30 rain later total RNA collected and analyzed by DDA. Examination of DDA gels indicated that IEC-6 ceils expressed some 8000 mRNAs, 60 of which were differentially expressed in response to pntrescine. Sequencing of one of the 60 differentially expressed amplicons yielded a message fragment composed of 295 bases (put 18). Put 18 displayed 100% homology over the 82 bases which represent the consensus sequence of the non translated BC-1 small RNA, the transcript of the genomic identifier sequence (ID seq). A second fragment (put 14) yielded a similar result; an mRNA composed of 253 bases, 82 of which displayed 100% homology with the consensus sequence of the BC-1 RNA. The ID sequence is represented in the rat genome between 1 and 1.5 x 105 times and appears in both 5' flanking and intronie sequences of genes coding for numerous growth related proteins including calmodulin III, prepro EGF, ODC, GH and c-Ha-ras. In addition, the BC-I transcript appears in a number of the mRNAs coding for these same proteins. We hypothesize that polyamines regulate the expression of the large number of ID sequence-containing genes by modulating transcription of the ID seq and expression of transcripts which contain a sequence similar to the BC-1 RNA. These data suggest that hormones such as CCK, IGF-1 and gastrin which induce the enzyme ornithine decarboxylase to catalyze putrescine synthesis act in part by modulating expression of ID seq containing genes. Supported in part by NIH DK 34110, NATO and the Philippe Foundation.
• IN S1TU H Y B R I D I Z A T I O N O F THE GLP-1 (7-36)NH 2 R E C E P T O R IN RAT GASTRIC M U C O S A L CELLS. W. Schepp, *S. Brandl, *]3. Pfitz, K. Dehne, I. Schmidfler, C. Prinz, V. Schusdziarra, M. Classen, and *H. Htfler,. Departments of Internal Medicine II and *Pathology, Technical University of Munich, Germany. We have previously shown that the intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36)NH 2 stimulates H + production in isolated rat partietal cells (Am. J. Physiol. 1991;260:G940-50 and 1994;266: G775-82). However, with Northern blot analysis we detected the mRNA of the GLP-1 (7-36)NH 2 receptor not only in parietal ceils, but also in a distinct population of non-parietal cells with small diameter (Am. J. Physiol. 1994;267:G423-32), a fraction known to contain endocrine cells. In oar present study we used in situ hybridization in combination with immunocytochemistry to further characterize GLP-1 receptor-positive cells. Enzymaticall.y isolated rat gastric mucosai cells were separated by counterflow elutnation into a fraction of small cells (diameter = 8-12 gin; parietal cell content 3-5%; FI/F2) and in a fraction enriched in parietal cells (80%; cell diameter 16-18 gin; FS). Cytospins of F1-F5 cells were fixed with 4% paraformaldehyde/PBS and incubated overnight at 52 ° C with the [35S]qabeled specific GLP-1 receptor probe prepared from the eDNA of a rat pancreatic islet eDNA library. After hybridization cells were washed at high stringency at 52 ° C, followed by immunocytochemistry for somatostatin (mouse anti-somatostatin antibody, Novo Wak, Bagsvaerd, Denmark, dilution 1:500), detected by avidin-biotin complex (Vector, Burlingame) and diaminobenzidin staining. Thereafter, the slides were covered by Kodak NTB2 film emulsion, followed by 14-28 days of autoradiography and counterstaining with hematoxilin/eosin. In all parietal cells of F5, in situ hybridization of the GLP-1 (7-36)NH 2 receptor mRNA was detectable after 14 days and pronounced after 28 days of autoradiography. However, in small cells (F1/F2) in situ hybridization of the GLP-1 (7-36)NH 2 receptor mRNA was pronounced already after 14 days of autoradiography. This signal was detected in 15-20% of F1/F2 cells. Immunocytochemically the F1/F2 cells with positive in situ hybridization did not stain positive for somatostatin and thus could not be identified as D-cells which represented a 10-15% GLP-1 receptor mRNA-negative portion of F1/F2. In accordance with this result GLP-1 (7-36)NH 2 (10-It-10 -B M) failed to significantly stimulate somatostatin release from primary cultures of F1/F2 grown for 48 h (30-40% somatostatin-positive cells). We conclude that in the rat gastric mucosa receptors for GLP-1 (7-36)NH 2 are located on parietal cells and on a distinct population of cells with small diameter which, however, are not identical with D-cells. Further immunocytochemical studies will have to clarify whether these cells are tteurcendocrine or immunocompetent cells. Supported by DFG grants Sche 229/7-1, PR 411/2-1.
Hormones
and
Receptors
A1003
PACAP-INDUCED GROWTH OF RAT PANCREATIC ACINAR TUMOR CELLS INVOLVES INCREASED EXPRESSION OF CFOS AND CJUN AND ACTIVATION OF THE AP1 TRANSCRIPTION FACTORCOMPLEX. H.SchafeL LZhong, R.Gtinther, F.Gundlach, and W.E.Schmidt. Laboratory of Molecular Gastroenterology, Dept. of Medicine, ChristianAlbrechts-University of Kiel, Germany The recently discovered peptide hormone Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) exerts many biological actions on various tissues including brain, intestinal muscles and pancreas. Beside its role as secretagogue and neuromodulatory peptide, PACAP has been shown to promote cell growth. In this study, the mechanisms of the growth promoting activity of PACAP on the rat pancreatic acinar tumor cell AR4-2J were characterized. Methods: Growth of AR4-ZI cells was analyzed by means of [3H]thymidine incorporation. Expression of cfos and cjun in AR4-ZI cells was investigated by quantitative RT-PCR. Activation of the mature transcription factor AP1 was demonstrated using gel retardation assays of nuclear extracts from AR4-ZI cells. Results: Cell growth of AR4-ZI cells was maximally stimulated by PACAP[127] and PACAP[I-38] at a dose of I-I0 nM. As shown by RT-PCR, the mRNA level of cfos was more than 100-fold increased in response to PACAP (10 nM), whereas the mRNA level of cjun was only 5-fold elevated. Expression of cfos and cjun was rapidly (10-20 rain), transienlly and dose dopendentiy stimulated upon PACAP-1 receptor occupation. Compared to PACAP[1-38], PACAP[t27] was 20-50 fold less potent and VIP (1 pM) revealed no effect. A variety of stimulants i.e. dBrcAMP (10 p_M), forskolin (10 pM) or PMA (100 ng/ml) revealed lower effects compared to PACAP[1-38], even if administered simultaneously. The strongly stimulating effect of PACAP[1-38] (10 nM) on cfos and cjun expression was abolished in the presence of the PACAP-specific antagonist PACAP [6-38] (5 pM), the calmodulin inhibitor W7 (10 pM), the protein kinase C inhibitors H7 (10 phl), staurosporine (0,1 JaM) and calphostin (20 phi) or the tyrosine ldnase inhibitor genlstein (5 ~4). Gelretardation assays revealed activation of API by PACAP[1-27] and PACAP[1-38] in an equipotent manner but not by VIP or seeretin. Activation of AP] was less rapid and persisted for several hours. Conclusions: PACAP[1-38] and to a lesser extent PACAP[1-27] are capable of stimulating cell growth via interaction with the PACAP-1 receptor. This effect includes the activation of the transcription factor AP1 and the devation of mRNA levels of cfos and cjun. This elevation seems to be dependent on several upstream signals involving the actions of PKC, calmodulin and tyrosine kinases. (Supported by a grant of the DFG; Schm 805/4-1)
DIFFERENTIALEFFECTSOF SUBCUTANEOUSGLP-1 ON GASTRIC EMPTYING,INSULIN RELEASE AND EXOCRINE PANCREATIC SECRETION IN MAN. J. Schirra, M. I(utschinski, P. Kuwert, U. Wank, R. Arnold, B. GGke.Dept. of Gastroenterology,University Hospital of Marburg, Germany. Truncated glucagon-like peptide-1 (GLP-1)is a promising new tool in the therapy of type II-diabetas since it reduces postprandial glycemie (Digestion 1993;54:389-390). However, the relative impor. tance of retarded gastric emptying and enhancedinsulin release with GLP-1 remain to be elucidated. Therefore, this study assessedpostprandial gastric emptying pattern, exesrine pancreatic secretion, insulin release and plasma glucosein responseto subcutaneousGLP-I in the same experimental set-up. METHODS: 8 healthy male volunteers were studied on 3 separate days in random order. Following a 30 min basal period, 0.125 or 0.25 nmol/kg GLP-1 or saline were subcutaneously injected. 5 rain later, a mixed liqid meal (54% carbohydrate, 42% lipid, 4% protein) was orally ingested, Antroduodeaal motility (2 antral, 4 duodenal portsJ, gastric emptying (double marker dilution technique according to Gestroeuterotogy 1976;70:203-210), duodenal outputs of amylase and trypsin, plasma glucose from arterialized venous blood ("heated hand") and IR-Insulin were followed for t80 rain. Position of the duodenal probe across the pylorus was verified by measuringtransmucosalpotential difference, Rate constants Ik) and slopes (B) of the emptying curves of the meal marker PEG 4000 were calculated using a power exponential fit. RESULTS: Mean±SEM, *: p < 0.05 vs. saline, #: p < 0.05 vs. 0.125 nmol/kg GLP-1. Insul.: iesulinogenic Parameter Saline 0.125 nmollkg GLP-1 0.25 nmul/kg GLP-1 k 0.04±0,023 0.04±0.004 0.04_+0.0016 B 3.73±0.69 8.17±1.80" 16.44±3.23"# Lag period (rain) 31.8 _+4.5 46.6±5.3* 61.6±5.0*# 50% retention (rain) 44.4_+4,5 5G.6_+5,7" 71.1-+4.8"# 1% retention (rain) 153,5 +_9,9 156.8 ± 12.7 170.9 _+5.9 Time to phase I]1 (rain) 159,1_+8.0 145.3_+7.1 170.8±12.7 Amylase (kU;AUC 0-!20 rain) 24.8_+8.4 23.9 ±6 23.4±5.3 Amylase (kU;AUC 0-30 min) 6.3± 1.9 3,8± 1.7" 1.0±0.7"# Amylase (kU;AUC 30-60 min) 5.4-+2.3 8.6_+2.2* 9.7± 1.8"# Glucese (mmol/l.f 80 rain) 15.3_+3.0 8.9±1.2" 7.3+_2.0* Insulin (mU/l.180 r a i n ) 188,6±27.0 331.7±68.8" 402.7_+85.8* InsuL index (mU/mmol'180 min) 12.4_+9.5 53.1±12.6" 70.3+_17.1" Total plesma glucoseresponse (AUC over besal.180 rain) was inversely correlated with total insulin releese (r=0.52; p
1995
• PUTRESCINE STIMULATES TRANSCRIPTION OF mRNAs CONTAINING THE BC-1 S E Q U E N C E . . J - L Scemama*. V. Rajah. P Brachet and ER Seidel. Depts of Physiology and Biology*, East Carolina University School of Medicine, Greenville NC 27858. The potyamine putrescine is imimately involved in regulation of mucosal growth, however the molecular mechanism by which it exerts control over proliferation remains unknown. We have employed the differential display assay (DDA) to examine polyamine-regulated gene transcription. IEC-6 cells were plated in DMEM containing 5 mM difluoromethylornithine (DFMO). After 72 hr, 10 ktM pntrescine was added to one half of the culture plates and 30 rain later total RNA collected and analyzed by DDA. Examination of DDA gels indicated that IEC-6 ceils expressed some 8000 mRNAs, 60 of which were differentially expressed in response to pntrescine. Sequencing of one of the 60 differentially expressed amplicons yielded a message fragment composed of 295 bases (put 18). Put 18 displayed 100% homology over the 82 bases which represent the consensus sequence of the non translated BC-1 small RNA, the transcript of the genomic identifier sequence (ID seq). A second fragment (put 14) yielded a similar result; an mRNA composed of 253 bases, 82 of which displayed 100% homology with the consensus sequence of the BC-1 RNA. The ID sequence is represented in the rat genome between 1 and 1.5 x 105 times and appears in both 5' flanking and intronie sequences of genes coding for numerous growth related proteins including calmodulin III, prepro EGF, ODC, GH and c-Ha-ras. In addition, the BC-I transcript appears in a number of the mRNAs coding for these same proteins. We hypothesize that polyamines regulate the expression of the large number of ID sequence-containing genes by modulating transcription of the ID seq and expression of transcripts which contain a sequence similar to the BC-1 RNA. These data suggest that hormones such as CCK, IGF-1 and gastrin which induce the enzyme ornithine decarboxylase to catalyze putrescine synthesis act in part by modulating expression of ID seq containing genes. Supported in part by NIH DK 34110, NATO and the Philippe Foundation.
• IN S1TU H Y B R I D I Z A T I O N O F THE GLP-1 (7-36)NH 2 R E C E P T O R IN RAT GASTRIC M U C O S A L CELLS. W. Schepp, *S. Brandl, *]3. Pfitz, K. Dehne, I. Schmidfler, C. Prinz, V. Schusdziarra, M. Classen, and *H. Htfler,. Departments of Internal Medicine II and *Pathology, Technical University of Munich, Germany. We have previously shown that the intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36)NH 2 stimulates H + production in isolated rat partietal cells (Am. J. Physiol. 1991;260:G940-50 and 1994;266: G775-82). However, with Northern blot analysis we detected the mRNA of the GLP-1 (7-36)NH 2 receptor not only in parietal ceils, but also in a distinct population of non-parietal cells with small diameter (Am. J. Physiol. 1994;267:G423-32), a fraction known to contain endocrine cells. In oar present study we used in situ hybridization in combination with immunocytochemistry to further characterize GLP-1 receptor-positive cells. Enzymaticall.y isolated rat gastric mucosai cells were separated by counterflow elutnation into a fraction of small cells (diameter = 8-12 gin; parietal cell content 3-5%; FI/F2) and in a fraction enriched in parietal cells (80%; cell diameter 16-18 gin; FS). Cytospins of F1-F5 cells were fixed with 4% paraformaldehyde/PBS and incubated overnight at 52 ° C with the [35S]qabeled specific GLP-1 receptor probe prepared from the eDNA of a rat pancreatic islet eDNA library. After hybridization cells were washed at high stringency at 52 ° C, followed by immunocytochemistry for somatostatin (mouse anti-somatostatin antibody, Novo Wak, Bagsvaerd, Denmark, dilution 1:500), detected by avidin-biotin complex (Vector, Burlingame) and diaminobenzidin staining. Thereafter, the slides were covered by Kodak NTB2 film emulsion, followed by 14-28 days of autoradiography and counterstaining with hematoxilin/eosin. In all parietal cells of F5, in situ hybridization of the GLP-1 (7-36)NH 2 receptor mRNA was detectable after 14 days and pronounced after 28 days of autoradiography. However, in small cells (F1/F2) in situ hybridization of the GLP-1 (7-36)NH 2 receptor mRNA was pronounced already after 14 days of autoradiography. This signal was detected in 15-20% of F1/F2 cells. Immunocytochemically the F1/F2 cells with positive in situ hybridization did not stain positive for somatostatin and thus could not be identified as D-cells which represented a 10-15% GLP-1 receptor mRNA-negative portion of F1/F2. In accordance with this result GLP-1 (7-36)NH 2 (10-It-10 -B M) failed to significantly stimulate somatostatin release from primary cultures of F1/F2 grown for 48 h (30-40% somatostatin-positive cells). We conclude that in the rat gastric mucosa receptors for GLP-1 (7-36)NH 2 are located on parietal cells and on a distinct population of cells with small diameter which, however, are not identical with D-cells. Further immunocytochemical studies will have to clarify whether these cells are tteurcendocrine or immunocompetent cells. Supported by DFG grants Sche 229/7-1, PR 411/2-1.
Hormones
and
Receptors
A1003
PACAP-INDUCED GROWTH OF RAT PANCREATIC ACINAR TUMOR CELLS INVOLVES INCREASED EXPRESSION OF CFOS AND CJUN AND ACTIVATION OF THE AP1 TRANSCRIPTION FACTORCOMPLEX. H.SchafeL LZhong, R.Gtinther, F.Gundlach, and W.E.Schmidt. Laboratory of Molecular Gastroenterology, Dept. of Medicine, ChristianAlbrechts-University of Kiel, Germany The recently discovered peptide hormone Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) exerts many biological actions on various tissues including brain, intestinal muscles and pancreas. Beside its role as secretagogue and neuromodulatory peptide, PACAP has been shown to promote cell growth. In this study, the mechanisms of the growth promoting activity of PACAP on the rat pancreatic acinar tumor cell AR4-2J were characterized. Methods: Growth of AR4-ZI cells was analyzed by means of [3H]thymidine incorporation. Expression of cfos and cjun in AR4-ZI cells was investigated by quantitative RT-PCR. Activation of the mature transcription factor AP1 was demonstrated using gel retardation assays of nuclear extracts from AR4-ZI cells. Results: Cell growth of AR4-ZI cells was maximally stimulated by PACAP[127] and PACAP[I-38] at a dose of I-I0 nM. As shown by RT-PCR, the mRNA level of cfos was more than 100-fold increased in response to PACAP (10 nM), whereas the mRNA level of cjun was only 5-fold elevated. Expression of cfos and cjun was rapidly (10-20 rain), transienlly and dose dopendentiy stimulated upon PACAP-1 receptor occupation. Compared to PACAP[1-38], PACAP[t27] was 20-50 fold less potent and VIP (1 pM) revealed no effect. A variety of stimulants i.e. dBrcAMP (10 p_M), forskolin (10 pM) or PMA (100 ng/ml) revealed lower effects compared to PACAP[1-38], even if administered simultaneously. The strongly stimulating effect of PACAP[1-38] (10 nM) on cfos and cjun expression was abolished in the presence of the PACAP-specific antagonist PACAP [6-38] (5 pM), the calmodulin inhibitor W7 (10 pM), the protein kinase C inhibitors H7 (10 phl), staurosporine (0,1 JaM) and calphostin (20 phi) or the tyrosine ldnase inhibitor genlstein (5 ~4). Gelretardation assays revealed activation of API by PACAP[1-27] and PACAP[1-38] in an equipotent manner but not by VIP or seeretin. Activation of AP] was less rapid and persisted for several hours. Conclusions: PACAP[1-38] and to a lesser extent PACAP[1-27] are capable of stimulating cell growth via interaction with the PACAP-1 receptor. This effect includes the activation of the transcription factor AP1 and the devation of mRNA levels of cfos and cjun. This elevation seems to be dependent on several upstream signals involving the actions of PKC, calmodulin and tyrosine kinases. (Supported by a grant of the DFG; Schm 805/4-1)
DIFFERENTIALEFFECTSOF SUBCUTANEOUSGLP-1 ON GASTRIC EMPTYING,INSULIN RELEASE AND EXOCRINE PANCREATIC SECRETION IN MAN. J. Schirra, M. I(utschinski, P. Kuwert, U. Wank, R. Arnold, B. GGke.Dept. of Gastroenterology,University Hospital of Marburg, Germany. Truncated glucagon-like peptide-1 (GLP-1)is a promising new tool in the therapy of type II-diabetas since it reduces postprandial glycemie (Digestion 1993;54:389-390). However, the relative impor. tance of retarded gastric emptying and enhancedinsulin release with GLP-1 remain to be elucidated. Therefore, this study assessedpostprandial gastric emptying pattern, exesrine pancreatic secretion, insulin release and plasma glucosein responseto subcutaneousGLP-I in the same experimental set-up. METHODS: 8 healthy male volunteers were studied on 3 separate days in random order. Following a 30 min basal period, 0.125 or 0.25 nmol/kg GLP-1 or saline were subcutaneously injected. 5 rain later, a mixed liqid meal (54% carbohydrate, 42% lipid, 4% protein) was orally ingested, Antroduodeaal motility (2 antral, 4 duodenal portsJ, gastric emptying (double marker dilution technique according to Gestroeuterotogy 1976;70:203-210), duodenal outputs of amylase and trypsin, plasma glucose from arterialized venous blood ("heated hand") and IR-Insulin were followed for t80 rain. Position of the duodenal probe across the pylorus was verified by measuringtransmucosalpotential difference, Rate constants Ik) and slopes (B) of the emptying curves of the meal marker PEG 4000 were calculated using a power exponential fit. RESULTS: Mean±SEM, *: p < 0.05 vs. saline, #: p < 0.05 vs. 0.125 nmol/kg GLP-1. Insul.: iesulinogenic Parameter Saline 0.125 nmollkg GLP-1 0.25 nmul/kg GLP-1 k 0.04±0,023 0.04±0.004 0.04_+0.0016 B 3.73±0.69 8.17±1.80" 16.44±3.23"# Lag period (rain) 31.8 _+4.5 46.6±5.3* 61.6±5.0*# 50% retention (rain) 44.4_+4,5 5G.6_+5,7" 71.1-+4.8"# 1% retention (rain) 153,5 +_9,9 156.8 ± 12.7 170.9 _+5.9 Time to phase I]1 (rain) 159,1_+8.0 145.3_+7.1 170.8±12.7 Amylase (kU;AUC 0-!20 rain) 24.8_+8.4 23.9 ±6 23.4±5.3 Amylase (kU;AUC 0-30 min) 6.3± 1.9 3,8± 1.7" 1.0±0.7"# Amylase (kU;AUC 30-60 min) 5.4-+2.3 8.6_+2.2* 9.7± 1.8"# Glucese (mmol/l.f 80 rain) 15.3_+3.0 8.9±1.2" 7.3+_2.0* Insulin (mU/l.180 r a i n ) 188,6±27.0 331.7±68.8" 402.7_+85.8* InsuL index (mU/mmol'180 min) 12.4_+9.5 53.1±12.6" 70.3+_17.1" Total plesma glucoseresponse (AUC over besal.180 rain) was inversely correlated with total insulin releese (r=0.52; p