- Email: [email protected]
gastrin receptor induces cell cycle progression in swiss 3T3 cells
AR42J IEC-6
CCKB-R Sites/Cel I
GP-R SiteslCel I
19,936+_ 3,245 <200/cell
t,146+_ 339 1,246+ 317
*RBA of G-17 for CCKB-R GP-R
80+ 15% NA
<0.01 76 + 11
2585
1Growthresponseto G-17 rhPO
45.0+_ 9t 80 + 11
Angiotensin II Is a Potent Growth Factor for IEC-18 Cells. Terence T. Chiu, Chintda Santiskulvong, Enrique Rozengurt, UCLA School of Medicine, Los Angeles, CA
100 1O0
*RBAof CCK-8and PG arbitrarilyassignedat 100%for CCKB-Rand GP-R,respec~vely. ?Growthresponseto lnM rhPGassigned100%value, rhPGincreasedgrowthof AR42Jand IEC6 cells by -225 and -320% abovecontrollevels,raspectJvely. :[:p<.05vs rhPGgrowtheffects. NA=notapplicabl 2583 Activation Of CCKe/Gastrin Receptor induces Cell Cycle Progression In Swiss 31"3 Cells. Elena Zhukova, James Sinnett-Smith, Angela Guiles, Helen Wong, Steve Young, John H. Walsh, Enrique Rozengurt, UCLA, Los Angeles, CA
Background. The CCKs/gastrin receptor plays an important role in the regulation of oastric cell proliferation in vivo. A number of cellular model systems have been utilized to unravel the signal traneduction pathways activated by CCK and gastrin via CCl(e/gectrin receptor in vitro, but a high-efficiancysystem has not, as yet, beendeveloped.Thus, until now, the effect of gastrin and CCKon the activation of the cell cycle machineryof the cell, including accumuietion of cyclins, regulation of cyclin-dependentkinases and the down-regula~on of cell cycle inhibitors, remainedentirely unexplored.Methodsand results. A retroviral genetransfer system was used to stably co-transfect CCKe/gastrinreceptor and Green Ruorascent Protein (GFP) in Swiss 313 cells. After GFP sorting, intracelluier Ca2÷ responsesto CCK were obtained in greater than 99% of cells, compared to 75-90% before GFP sorting. Scatchard analysis showed that transfected receptor binds '~I-CCK8 with a Kd= 1.1riM. Agonict-acthration of the CCKe/gastrinreceptor induced a dose-dependentactivation of ERK-2 and stdldog dosedependent stimulation of DNA synthesis in transfeeted Swiss 3"13 cells. In the presence of insulin (1/,~g/m/), addition of 50 nM CCK or gestrin induced a 66.5 + 8.8 (mean _+ SEM, n = 24 in 8 independentexperiments)fold increasein cellular DNA synthesis, reachinga level similar to that achieved by stimulation with a saturating concentration of fresh serum. The stimulation induced by gastrin and insulin was much greater than the responseto satureting concentrations of either gastrin or insulin given separately.The large transition from G~to S phase induced by CCKe/gastrinreceptoractivation in Swiss 3T3 cells incubatedin the presence of insulin, is associated with a striking increase in the expression of cyclins D1, D3 and E, downregulationof p27K%ndhyperphosphorylationof Rb. Similar effects were observedwhen CCKe/gastrinreceptor was activated in the presenceof EGF (5 ng/ml) or bombasio (10 riM). Conclusions. Our results demonstratethat activation of CCKe/gastrinreceptor in combination with insulin, EGF or bombesin receptors induces a potent synergistic effect on progression through the cell cycle. CCK~gastrinreceptor,retrovirally-transfectedinto Swiss 3T3 cell, provides an excellent model to study mitogenic signal transduction and cell cycle actk,ation induced by CCK and gastrin. 2584
Cellular Localization of CCK-BRi4sv in Human Coloroctal Cancers Xian-Liang Rui, Helen L. Hellmich, R.y. Declan Fleming, Courtney M. Townsend Jr., Mark R. Hellmich, Univ of Texas Medical Branch, Galveston,1)( BACKGROUND.Recentlywe have isolated and partial characterized a novel splice variant of the human cholecystokinin-B/gastrin (CCK-B) receptor. The splice variant, designated CCKBRi4sv is generated by intron 4 retention, which results in a 69 amino acid insertion in its third intracellularloop domain. Expressionstudies haverevealedthat CCK-BRi4svbinds gastrin (G-17) and is coupled to the regulation of intrecellular Ca2+ ([Ca2*],) through both liganddependent and -independent pathways. Although RT-PCR and RNAse protection assays showed that CCK-BRi4svwas selectively expressed in colorectal cancers and adenomatous polyps, but not by the normal colonic mucosa, the precise identity of the cells expressing CCK-BRi4svwithin these tissues were not determined.The aim of this study was to identify the cell type(s) expressingCCK-BRi4svin freshly resectedhuman coiorectal cancers. METHODS. Human colorectal cancers were collected immediately following resection, For intracellular Ca2. measurements,the tissue was dissociated with collagenase(1000 U/ml for 90 min at 37C) and the dispersed cells plated onto coverslips and load with the Ca2+ indicator dye, Fura-2 (2/~M). For in situ hybridization the tissue was fixed in 4% paraformaldebyde(2 h at 4C) and sectioned on a cryostat microtome. Immunostainiog with anti-cytokeratin and vimentin antibodies was used to identify cells as either epithelial or mesenchymal origin, respectively.RESULTS.Seventeencancerspecimenswere analyzedfor G-17-inducedincreases in [Ca2÷],.G-17 responsive cells were identified in 71% of the specimens. In 83% of the G17 responsive cultures, the cells that showed increased [Ca2+], also stained positive for cytokerstin. In four cultures, cell that stained positive for vimentin exhibited G-17 induced increases in [Ca2+],. /n situ hybridization, using a radioiebel cRNA probe specific for CCKBRi4sv, revealed hybridization in epithelial cells of the tumor tissue. CONCLUSION.In the majority of human colorectal cancers specimens studied, epithelial cells respondedto G-17 stimulation with an increase in [Ca2+],. In situ hybridization revealedthat CCK-BRi4svmRNA expression also occurred in epithelial cells, suggesting that the G-17 regulation of [Ca2+],in these cells is mediated by CCK-BRi4sv.
A-508
Background/Aims: Non-transtormnd epitheliel IEC-18 cells, originally derived from fetal rat ileal crypts, have served as a model for intestinal cell migretton, restitution, growth and differentiation. Recently, we reported that angiotansin II (ANG II) can induce rapid calcium mobilization and striking protein IonaseO (PKD) activation through a protein kinaseC (PKC)dependentpathway. However, it is not known whether ANG II can act as a growth factor for epithelial intestinal cells. Here, we examined whether ANG II stimulates extracellular signalregulated protein kinases 1 and 2 (ERK1/2) activation, ONA synthesis and cell proliferation in IEC-18 cells. Results: Treatment of sarum-deprived IEC-18 cells with ANG it (100 nM) led to a rapid and striking activation of ERK1/2, which can be inhibited by the selective AT1 receptor antagonist Losartan (lpM). A detectableeffect was obtained within 15 sec of ANG II treatment and ma,dmal effect was seen after 2.5 rain of incubation. ERK1/2 activation induced by ANG II was completed blockedby treatment (lh) with the selectiveMEK inhibitors, PD98059 (lOpM) and U0126 (2.5/~d). We also demonstratedthat ANG II, via AT1 receptor, induces a sldldng increasein DNA synthesis in IEC-18 cells as judged by measuringtritiatedthymidina incorporation. A detectableeffect was obtained with 0.1 nM ANG II and maximal effect (4 fold) was achievedat 10 nM. In contrast, EGF (5ng/ml) led to only 1.6 fold increase. Furthermore,ANG II induceda doubling in cell number within 24 h of stimulation. Pretreatment of cell cultures with the MEK inhibitors, P098059 (IO/~M) and U0126 (2.5/~M), also attenuated (50%) ANG IHnduced trftiated-thymidine incorporation. Conclusion: Our results demonstrate that ANG II, via ATf receptor, acts as a potent growth factor in IEC-18 cells and induces a rapid and dramatic ERK1/2 activation. ANG II-induced DNA synthesis is partially dependent on ERK1/2 activation, indicating that the mitogenic response induced by ANG li is mediated by E R K - d e l ~ t and ERK-independentpathways. 25416
ExpraceiN of tile Human Trefoil Factor 3 (TFF3) in Relation to Growth and 01ffof~iofion of HT-29 Coil Soflpopulofiona. Stephane Ourual, Frederic Moro, INSERM U45, Lyon France; Valerie Gouyer, INSERM U377, Ulle France; Carina Bienchard, INSERM U45, Lyon France; Christian L Laboisse, INSERM U539, Nantes France; Jean-PierreAubart, INSERM U377, Lille France;Andrew Giraud, Oept of Medicine, Melbourne Only, Melbourne Austratla; Jean-Cieude Cuber, INSERM LI45, Lyon France Background : Trefoil peptidec 1 and 2 (TFF1 and 2) are expressed in goblet cells of the stomach whereasTFF3 is localized in mucus cells of the intestine. In the present study, TFF3 expression was analyzed in relation to cell growth, in parental HT-29 cells and in several populations of subciones including the HT-29 16E cell line and a clone selected by adaptation to methotrexate (HT-29 MTX). Both cell lines exhibit a phenotype of mucus-secrating cells. Additionally, dedifforontiated HT-29 MTX cells which constitutively express UEV (ubiquitinconjugatingE2 enzymevariant) andthe cional cell line HT-2919A, which harbors an enterocytelike type were also investigated.Methods:Theexpressionof TFF3during growth and differentiatton was evaluated by RT-PCR, Northern blot and radioimmunoassay.The nature of the immunoreaetive material was revealed by Western blot and gel chromatography.Results:in the parental population, TFF3 levels were very low even at late confluency. In contrast, the expressionof TR:3 which was barely detectablein the clone 16E early after seeding gradually increased till confluency and reached a maximal level two weeks later. Thereafter, expression of TFF3 remainedat the very high level till the end of the 55 days experimentalperiod. Overall, a lO-fold increase in TFF3-cellcontent was observed betweenthe early proliferative state and the late contluency. In contrast, TR:I and TFF2were barely detectableduring the cell growth and differentiation period. The TFFI-cell content was about lO0-foid lower as compared to that of TFF3. The levels of MUC 2 and MUC 3 mRNAs also increased in the course of the differentiation process. TFF3 was specifically localized within mucus vacuoles by contocal microscopy, The HT-29 MTX cells also expressed high levels of ]TF3 in the late confiuency. In contrast, the dedifferenttatedHT-29 MTX-UEVcells and the enterocyte-likecell line HT-29 19A or a variety of other intestinal cell lines were shown to contain low levels of TFF3 immunoreactivity. Conclusion:Together,these data indicate that TFF3 expression is dramaticall'/increased in the course of the differentiation process of the mucus secreting cell lines derived from the parentalHT-29 cells. Overall,these mucus-producingcells representa useful tool to study the transcriptional activation of the TFF3 gene in relation to differentiation of goblet cells and to investigatethe mechanismsunderlyingthe secretoryprocessof this peptide.
2587 IGFRP-3 Interacts wlib Type V TSF-.8 Receptor to Activate Smad2 and Inhibit Growth of Human Intestinal Smooth Muscle. John F. Kuemmerie, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND:We have previously shown that human intestinal muscle cells secrete IGF-I, IGF binding protein-3 (IGFBP-3), and TGF-#1. Their pattern of secretion during growth in cuifure parallels their effects on growth: IGF-Jsecretion and its ability to promote growth are highest during the period of rapid growth, whereas IGFBP-3 and TGF-#1 secretion and the ability of TGF-#1 to inhibit growth are highest after confluence. IGFBP-3 has been shown to bind Type V TGF-8 receptor in various cell lines (JBC 272:20572,1997). AIM: To determine whether IGFBP-3 hinds to Type V TGF-p receptor in human intestinal smooth muscle, and to identify its signaling pathways and effects on cell growth. METHODS: Intestinal smooth muscle cells were isolatedfrom the circular layer of human jejunum, cultured in DMEM+ 10% F{;S and used in first passage.The direct, IGF-I independenteffects of IGFBP-3were identified in the presence of an IGF-I receptor antagonist, IGF analog. Growth was measured by the incorporation of [3H]thymidineafter 24h incubation in serum-free medium. Interaction of TGF#1 and IGFBP-3 with Type V TGF-/3 receptor was measured by ('~I]TGF-#1 affinity labeling and autoradiography.Activation of Smad2,the initial subatrateof the activatedTGF-/3receptor, was measured by Western blot analysis using an antibody to Ser465/467-phosphorylated
CCKB-R Sites/Cel I
GP-R SiteslCel I
19,936+_ 3,245 <200/cell
t,146+_ 339 1,246+ 317
*RBA of G-17 for CCKB-R GP-R
80+ 15% NA
<0.01 76 + 11
2585
1Growthresponseto G-17 rhPO
45.0+_ 9t 80 + 11
Angiotensin II Is a Potent Growth Factor for IEC-18 Cells. Terence T. Chiu, Chintda Santiskulvong, Enrique Rozengurt, UCLA School of Medicine, Los Angeles, CA
100 1O0
*RBAof CCK-8and PG arbitrarilyassignedat 100%for CCKB-Rand GP-R,respec~vely. ?Growthresponseto lnM rhPGassigned100%value, rhPGincreasedgrowthof AR42Jand IEC6 cells by -225 and -320% abovecontrollevels,raspectJvely. :[:p<.05vs rhPGgrowtheffects. NA=notapplicabl 2583 Activation Of CCKe/Gastrin Receptor induces Cell Cycle Progression In Swiss 31"3 Cells. Elena Zhukova, James Sinnett-Smith, Angela Guiles, Helen Wong, Steve Young, John H. Walsh, Enrique Rozengurt, UCLA, Los Angeles, CA
Background. The CCKs/gastrin receptor plays an important role in the regulation of oastric cell proliferation in vivo. A number of cellular model systems have been utilized to unravel the signal traneduction pathways activated by CCK and gastrin via CCl(e/gectrin receptor in vitro, but a high-efficiancysystem has not, as yet, beendeveloped.Thus, until now, the effect of gastrin and CCKon the activation of the cell cycle machineryof the cell, including accumuietion of cyclins, regulation of cyclin-dependentkinases and the down-regula~on of cell cycle inhibitors, remainedentirely unexplored.Methodsand results. A retroviral genetransfer system was used to stably co-transfect CCKe/gastrinreceptor and Green Ruorascent Protein (GFP) in Swiss 313 cells. After GFP sorting, intracelluier Ca2÷ responsesto CCK were obtained in greater than 99% of cells, compared to 75-90% before GFP sorting. Scatchard analysis showed that transfected receptor binds '~I-CCK8 with a Kd= 1.1riM. Agonict-acthration of the CCKe/gastrinreceptor induced a dose-dependentactivation of ERK-2 and stdldog dosedependent stimulation of DNA synthesis in transfeeted Swiss 3"13 cells. In the presence of insulin (1/,~g/m/), addition of 50 nM CCK or gestrin induced a 66.5 + 8.8 (mean _+ SEM, n = 24 in 8 independentexperiments)fold increasein cellular DNA synthesis, reachinga level similar to that achieved by stimulation with a saturating concentration of fresh serum. The stimulation induced by gastrin and insulin was much greater than the responseto satureting concentrations of either gastrin or insulin given separately.The large transition from G~to S phase induced by CCKe/gastrinreceptoractivation in Swiss 3T3 cells incubatedin the presence of insulin, is associated with a striking increase in the expression of cyclins D1, D3 and E, downregulationof p27K%ndhyperphosphorylationof Rb. Similar effects were observedwhen CCKe/gastrinreceptor was activated in the presenceof EGF (5 ng/ml) or bombasio (10 riM). Conclusions. Our results demonstratethat activation of CCKe/gastrinreceptor in combination with insulin, EGF or bombesin receptors induces a potent synergistic effect on progression through the cell cycle. CCK~gastrinreceptor,retrovirally-transfectedinto Swiss 3T3 cell, provides an excellent model to study mitogenic signal transduction and cell cycle actk,ation induced by CCK and gastrin. 2584
Cellular Localization of CCK-BRi4sv in Human Coloroctal Cancers Xian-Liang Rui, Helen L. Hellmich, R.y. Declan Fleming, Courtney M. Townsend Jr., Mark R. Hellmich, Univ of Texas Medical Branch, Galveston,1)( BACKGROUND.Recentlywe have isolated and partial characterized a novel splice variant of the human cholecystokinin-B/gastrin (CCK-B) receptor. The splice variant, designated CCKBRi4sv is generated by intron 4 retention, which results in a 69 amino acid insertion in its third intracellularloop domain. Expressionstudies haverevealedthat CCK-BRi4svbinds gastrin (G-17) and is coupled to the regulation of intrecellular Ca2+ ([Ca2*],) through both liganddependent and -independent pathways. Although RT-PCR and RNAse protection assays showed that CCK-BRi4svwas selectively expressed in colorectal cancers and adenomatous polyps, but not by the normal colonic mucosa, the precise identity of the cells expressing CCK-BRi4svwithin these tissues were not determined.The aim of this study was to identify the cell type(s) expressingCCK-BRi4svin freshly resectedhuman coiorectal cancers. METHODS. Human colorectal cancers were collected immediately following resection, For intracellular Ca2. measurements,the tissue was dissociated with collagenase(1000 U/ml for 90 min at 37C) and the dispersed cells plated onto coverslips and load with the Ca2+ indicator dye, Fura-2 (2/~M). For in situ hybridization the tissue was fixed in 4% paraformaldebyde(2 h at 4C) and sectioned on a cryostat microtome. Immunostainiog with anti-cytokeratin and vimentin antibodies was used to identify cells as either epithelial or mesenchymal origin, respectively.RESULTS.Seventeencancerspecimenswere analyzedfor G-17-inducedincreases in [Ca2÷],.G-17 responsive cells were identified in 71% of the specimens. In 83% of the G17 responsive cultures, the cells that showed increased [Ca2+], also stained positive for cytokerstin. In four cultures, cell that stained positive for vimentin exhibited G-17 induced increases in [Ca2+],. /n situ hybridization, using a radioiebel cRNA probe specific for CCKBRi4sv, revealed hybridization in epithelial cells of the tumor tissue. CONCLUSION.In the majority of human colorectal cancers specimens studied, epithelial cells respondedto G-17 stimulation with an increase in [Ca2+],. In situ hybridization revealedthat CCK-BRi4svmRNA expression also occurred in epithelial cells, suggesting that the G-17 regulation of [Ca2+],in these cells is mediated by CCK-BRi4sv.
A-508
Background/Aims: Non-transtormnd epitheliel IEC-18 cells, originally derived from fetal rat ileal crypts, have served as a model for intestinal cell migretton, restitution, growth and differentiation. Recently, we reported that angiotansin II (ANG II) can induce rapid calcium mobilization and striking protein IonaseO (PKD) activation through a protein kinaseC (PKC)dependentpathway. However, it is not known whether ANG II can act as a growth factor for epithelial intestinal cells. Here, we examined whether ANG II stimulates extracellular signalregulated protein kinases 1 and 2 (ERK1/2) activation, ONA synthesis and cell proliferation in IEC-18 cells. Results: Treatment of sarum-deprived IEC-18 cells with ANG it (100 nM) led to a rapid and striking activation of ERK1/2, which can be inhibited by the selective AT1 receptor antagonist Losartan (lpM). A detectableeffect was obtained within 15 sec of ANG II treatment and ma,dmal effect was seen after 2.5 rain of incubation. ERK1/2 activation induced by ANG II was completed blockedby treatment (lh) with the selectiveMEK inhibitors, PD98059 (lOpM) and U0126 (2.5/~d). We also demonstratedthat ANG II, via AT1 receptor, induces a sldldng increasein DNA synthesis in IEC-18 cells as judged by measuringtritiatedthymidina incorporation. A detectableeffect was obtained with 0.1 nM ANG II and maximal effect (4 fold) was achievedat 10 nM. In contrast, EGF (5ng/ml) led to only 1.6 fold increase. Furthermore,ANG II induceda doubling in cell number within 24 h of stimulation. Pretreatment of cell cultures with the MEK inhibitors, P098059 (IO/~M) and U0126 (2.5/~M), also attenuated (50%) ANG IHnduced trftiated-thymidine incorporation. Conclusion: Our results demonstrate that ANG II, via ATf receptor, acts as a potent growth factor in IEC-18 cells and induces a rapid and dramatic ERK1/2 activation. ANG II-induced DNA synthesis is partially dependent on ERK1/2 activation, indicating that the mitogenic response induced by ANG li is mediated by E R K - d e l ~ t and ERK-independentpathways. 25416
ExpraceiN of tile Human Trefoil Factor 3 (TFF3) in Relation to Growth and 01ffof~iofion of HT-29 Coil Soflpopulofiona. Stephane Ourual, Frederic Moro, INSERM U45, Lyon France; Valerie Gouyer, INSERM U377, Ulle France; Carina Bienchard, INSERM U45, Lyon France; Christian L Laboisse, INSERM U539, Nantes France; Jean-PierreAubart, INSERM U377, Lille France;Andrew Giraud, Oept of Medicine, Melbourne Only, Melbourne Austratla; Jean-Cieude Cuber, INSERM LI45, Lyon France Background : Trefoil peptidec 1 and 2 (TFF1 and 2) are expressed in goblet cells of the stomach whereasTFF3 is localized in mucus cells of the intestine. In the present study, TFF3 expression was analyzed in relation to cell growth, in parental HT-29 cells and in several populations of subciones including the HT-29 16E cell line and a clone selected by adaptation to methotrexate (HT-29 MTX). Both cell lines exhibit a phenotype of mucus-secrating cells. Additionally, dedifforontiated HT-29 MTX cells which constitutively express UEV (ubiquitinconjugatingE2 enzymevariant) andthe cional cell line HT-2919A, which harbors an enterocytelike type were also investigated.Methods:Theexpressionof TFF3during growth and differentiatton was evaluated by RT-PCR, Northern blot and radioimmunoassay.The nature of the immunoreaetive material was revealed by Western blot and gel chromatography.Results:in the parental population, TFF3 levels were very low even at late confluency. In contrast, the expressionof TR:3 which was barely detectablein the clone 16E early after seeding gradually increased till confluency and reached a maximal level two weeks later. Thereafter, expression of TFF3 remainedat the very high level till the end of the 55 days experimentalperiod. Overall, a lO-fold increase in TFF3-cellcontent was observed betweenthe early proliferative state and the late contluency. In contrast, TR:I and TFF2were barely detectableduring the cell growth and differentiation period. The TFFI-cell content was about lO0-foid lower as compared to that of TFF3. The levels of MUC 2 and MUC 3 mRNAs also increased in the course of the differentiation process. TFF3 was specifically localized within mucus vacuoles by contocal microscopy, The HT-29 MTX cells also expressed high levels of ]TF3 in the late confiuency. In contrast, the dedifferenttatedHT-29 MTX-UEVcells and the enterocyte-likecell line HT-29 19A or a variety of other intestinal cell lines were shown to contain low levels of TFF3 immunoreactivity. Conclusion:Together,these data indicate that TFF3 expression is dramaticall'/increased in the course of the differentiation process of the mucus secreting cell lines derived from the parentalHT-29 cells. Overall,these mucus-producingcells representa useful tool to study the transcriptional activation of the TFF3 gene in relation to differentiation of goblet cells and to investigatethe mechanismsunderlyingthe secretoryprocessof this peptide.
2587 IGFRP-3 Interacts wlib Type V TSF-.8 Receptor to Activate Smad2 and Inhibit Growth of Human Intestinal Smooth Muscle. John F. Kuemmerie, Medical Coil of Virginia, VCU, Richmond, VA BACKGROUND:We have previously shown that human intestinal muscle cells secrete IGF-I, IGF binding protein-3 (IGFBP-3), and TGF-#1. Their pattern of secretion during growth in cuifure parallels their effects on growth: IGF-Jsecretion and its ability to promote growth are highest during the period of rapid growth, whereas IGFBP-3 and TGF-#1 secretion and the ability of TGF-#1 to inhibit growth are highest after confluence. IGFBP-3 has been shown to bind Type V TGF-8 receptor in various cell lines (JBC 272:20572,1997). AIM: To determine whether IGFBP-3 hinds to Type V TGF-p receptor in human intestinal smooth muscle, and to identify its signaling pathways and effects on cell growth. METHODS: Intestinal smooth muscle cells were isolatedfrom the circular layer of human jejunum, cultured in DMEM+ 10% F{;S and used in first passage.The direct, IGF-I independenteffects of IGFBP-3were identified in the presence of an IGF-I receptor antagonist, IGF analog. Growth was measured by the incorporation of [3H]thymidineafter 24h incubation in serum-free medium. Interaction of TGF#1 and IGFBP-3 with Type V TGF-/3 receptor was measured by ('~I]TGF-#1 affinity labeling and autoradiography.Activation of Smad2,the initial subatrateof the activatedTGF-/3receptor, was measured by Western blot analysis using an antibody to Ser465/467-phosphorylated