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...inistration (42). While details of the mechanisms ofTGF-j3 action remain unclear, the mitogenic effects are thought to be indirect, involving induction of platelet derived growth factor A and B chain =-=(43)-=-. This laboratory has previously shown that the ability of retinoids to control the growth and inhibit the transformation of 1OT’/2 cells is directly correlated with their ability to up-regulate GJC (...

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...unctional communication with neighboring normal cells (7). Evidence of a more circumstantial nature also supports this hypothesis; for example, GJC is frequently absent between tumor and normal cells =-=(8)-=- and is inhibited by certain oncogenes and tumor promoters (9, 10). Retinoids act in many respects as antitumor promoters and are able to suppress neoplastic transformation and enhance growth control....

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...ated from human and porcine platelets, respectively, were a generous gift from Dr. Michael Spomn (NCI, Bethesda, MD). Cells and Culture Conditions. The C3H/1 OT’/2 mouse embryo fibroblastic cell line =-=(65)-=- was used throughout this study and was routinely maintained in DMEM in 5% calf serum (HyClone Laboratories, Inc., Logan, UT) and 25 jWml gentamicin (Tn Bio Labs, Inc., PA). For experiments, cells we...

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...naling receptors (36). Receptor types I and II (37) mediate the effects of TGF-; type I mediates effects upon extracellular matrix proteins, while growth inhibition is mediated via type II receptors =-=(38)-=-. The situation, however, is complex because the final cellular response to TGF-3 depends upon receptor availability as a result of interactions between receptor types I and II (32, 37). Here we have ...

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...ct experimental support for this hypothesis comes from observations that growth of transformed cells can be inhibited by the establishment ofgap junctional communication with neighboring normal cells =-=(7)-=-. Evidence of a more circumstantial nature also supports this hypothesis; for example, GJC is frequently absent between tumor and normal cells (8) and is inhibited by certain oncogenes and tumor promo...

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...ed previously; j31 appears more active in endothelial cells (61), lL-3-stimulated hematopoietic cells (62), and MCF-7 cells (63). In contrast, f32 is more potent in a Xenopus mesoderm induction assay =-=(64)-=- and the growth stimulation of fibroblasts (63). For a number of years, our laboratory has presented data that has both developed and strongly supported the Concept of growth regulation via the transm...

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... (20). TGF- is able to inhibit the growth of a mange of normal and transformed epithelial cells, both in vitro (14, 15) and in vivo (40). In contrast, TGF-f3 is mitogenic for many fibmoblastic cells =-=(16, 18)-=- and can induce fibrosis after local (41) or systemic administration (42). While details of the mechanisms ofTGF-j3 action remain unclear, the mitogenic effects are thought to be indirect, involving i...

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...eins, named in accordance with their predicted molecular weights, has been described in a wide variety oftissues. The extracellular and transmembrane domains of connexin proteins are highly conserved =-=(4)-=- with the major differences observed in the cytoplasmic COOH-terminal region. Received 1/i 1/94; revised 3/i 8/94; accepted 4/5/94. 1 This work was supported by Grant CA 39947 from the NIH (to J. S. B...

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...dence of a more circumstantial nature also supports this hypothesis; for example, GJC is frequently absent between tumor and normal cells (8) and is inhibited by certain oncogenes and tumor promoters =-=(9, 10)-=-. Retinoids act in many respects as antitumor promoters and are able to suppress neoplastic transformation and enhance growth control. In the lOT1h cell system, these cancer preventive effects are str...

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...mplex; activators of protein kinase A have been shown to increase Cx43 levels and GJC in 1 OT1h cells and other cell lines (53). In contrast, other agents which induce Cx43 phosphorylation [e.g., EGF =-=(54)-=- and 12-O-tetradecanoylphorbol-1 3-acetate decrease commun ication in fibroblasts (55)]. A likely explanation of these confusing results is that Cx43 has multiple sites, some or all of which may influ...

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...ion of ions and molecules up to 1000 daltons (2). Each channel is comprised of two connexons, one from each cell. The connexon is a hexagonal structure composed of 6 transmembrane proteins termed Cx4 =-=(3)-=-. A family of related connexin proteins, named in accordance with their predicted molecular weights, has been described in a wide variety oftissues. The extracellular and transmembrane domains of conn...

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...ntributed by daughter cells of those labeled in the first 24 h, this seems unlikely to account for all the increased labeling measured at 48 h because 10T1/2 cells have a doubling time of about 1 6 h =-=(49)-=- (for example, labeling after 10 ng/ml TGF-2: 24 h, 9.48%; 48 h, 33.24%). Therefore, cells are still entering DNA synthesis 48 h after TGF- treatment at a time when GJC had been elevated for at leas...

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...odudion Gap junctions are direct hydrophilic channels with an inner diameter of 1 6-20 A that connect adjacent cells (1) and which allow the passive diffusion of ions and molecules up to 1000 daltons =-=(2)-=-. Each channel is comprised of two connexons, one from each cell. The connexon is a hexagonal structure composed of 6 transmembrane proteins termed Cx4 (3). A family of related connexin proteins, name...

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...wth factors upon cell proliferation and differentiation are diverse (20). TGF- is able to inhibit the growth of a mange of normal and transformed epithelial cells, both in vitro (14, 15) and in vivo =-=(40)-=-. In contrast, TGF-f3 is mitogenic for many fibmoblastic cells (16, 18) and can induce fibrosis after local (41) or systemic administration (42). While details of the mechanisms ofTGF-j3 action remain...

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...redictions of the effects of TGF-3 which, depending upon cell type, can act both as a mitogen and as an inhibitor of proliferation are not so straightforward. TGF-3 has been reported to both reduce =-=(45)-=- or enhance (46) intercellular communication in normal rat kidney cells. In BALB/c 3T3 fibmoblasts, TGF-f3 is reported either to increase (46) or have no effect (47) upon communication. These conflict...

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...ame senine/threonine protein kinases (32-34). The type Ill receptor has also been cloned (35) and is a pnoteoglycan that acts to regulate the access of TGF-f3 to the type I and II signaling receptors =-=(36)-=-. Receptor types I and II (37) mediate the effects of TGF-; type I mediates effects upon extracellular matrix proteins, while growth inhibition is mediated via type II receptors (38). The situation, ...

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...in 1 OT1h cells and other cell lines (53). In contrast, other agents which induce Cx43 phosphorylation [e.g., EGF (54) and 12-O-tetradecanoylphorbol-1 3-acetate decrease commun ication in fibroblasts =-=(55)-=-]. A likely explanation of these confusing results is that Cx43 has multiple sites, some or all of which may influence junctional assembly and/or func(ion. Certainly, several serine residues are invol...

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...escribes a family of closely related polypeptide growth factors; three isoforms are found in mammals (TGF-f31-3; Refs. 21-23), a fourth (TGF-134) in the chicken (24), and a fifth (TGF-f35) in Xenopus =-=(25)-=-. All five proteins are produced as inactive complexes and are dissociated (by proteolysis, for example) to produce active molecules (26). The active molecules are Mr 25,000 homodimers, consisting of ...

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.... The situation with regard to serine/threonine phosphorylation of Cx43 is complex; activators of protein kinase A have been shown to increase Cx43 levels and GJC in 1 OT1h cells and other cell lines =-=(53)-=-. In contrast, other agents which induce Cx43 phosphorylation [e.g., EGF (54) and 12-O-tetradecanoylphorbol-1 3-acetate decrease commun ication in fibroblasts (55)]. A likely explanation of these conf...

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... TGF-f3 family of growth factors upon cell proliferation and differentiation are diverse (20). TGF- is able to inhibit the growth of a mange of normal and transformed epithelial cells, both in vitro =-=(14, 15)-=- and in vivo (40). In contrast, TGF-f3 is mitogenic for many fibmoblastic cells (16, 18) and can induce fibrosis after local (41) or systemic administration (42). While details of the mechanisms ofTGF...

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...pes I, II, and V, of molecular weights 53,000, 73,000, and 400,000, respectively, have been cloned (28-31) and ame senine/threonine protein kinases (32-34). The type Ill receptor has also been cloned =-=(35)-=- and is a pnoteoglycan that acts to regulate the access of TGF-f3 to the type I and II signaling receptors (36). Receptor types I and II (37) mediate the effects of TGF-; type I mediates effects upon...

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...ap junctions are involved in a variety of biological processes, the best documented being the rapid transfer of ionic signals required for the coordinated contraction of the heart and term myometnium =-=(5)-=-. In addition, gap junctions are considered to have a functional role in tissue homeostasis, in nutrient and excretory product transfer in the avasculan lens and cornea (1 ), and in the transmission o...

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... (P < 0.05) from control treatment. A B .‘ ‘ wA , 4. , , ‘ ,‘. , (.., !. :l i . .. .f..,. I.;,t, o A mange of cell surface proteins have been proposed as TGF receptors =-=(27)-=-. Receptor types I, II, and V, of molecular weights 53,000, 73,000, and 400,000, respectively, have been cloned (28-31) and ame senine/threonine protein kinases (32-34). The type Ill receptor has also...

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...e that proliferation induced by a growth factor under defined conditions has been accompanied by increased GJC. Previous reports of increased cellto-cell coupling accompanying increased proliferation =-=(50)-=- resulted from wounding. Retinoid-induced up-regulation of GJC in 10T#{189}cells involves the localization of Cx43 into membrane plaques and its extensive phosphorylation (1 2). Cx43 synthesized after...

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...31 (Fig. 5). Differences in the potencies of TGF- isoforms have been meported previously; j31 appears more active in endothelial cells (61), lL-3-stimulated hematopoietic cells (62), and MCF-7 cells =-=(63)-=-. In contrast, f32 is more potent in a Xenopus mesoderm induction assay (64) and the growth stimulation of fibroblasts (63). For a number of years, our laboratory has presented data that has both deve...

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...TGF-134) in the chicken (24), and a fifth (TGF-f35) in Xenopus (25). All five proteins are produced as inactive complexes and are dissociated (by proteolysis, for example) to produce active molecules =-=(26)-=-. The active molecules are Mr 25,000 homodimers, consisting of two Mr 12,000 chains linked by disulfide bonds (19). Table I Effects of TGF-f31 (A), TGF-f32 (B), and TGF-f31 + f32(C) upon gap junctiona...

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...dence of a more circumstantial nature also supports this hypothesis; for example, GJC is frequently absent between tumor and normal cells (8) and is inhibited by certain oncogenes and tumor promoters =-=(9, 10)-=-. Retinoids act in many respects as antitumor promoters and are able to suppress neoplastic transformation and enhance growth control. In the lOT1h cell system, these cancer preventive effects are str...

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...riod, during which time Cx43 levels and GJC were consistently elevated (Fig. 3C). Discussion The effects of the TGF-f3 family of growth factors upon cell proliferation and differentiation are diverse =-=(20)-=-. TGF- is able to inhibit the growth of a mange of normal and transformed epithelial cells, both in vitro (14, 15) and in vivo (40). In contrast, TGF-f3 is mitogenic for many fibmoblastic cells (16, ...

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...1 9 and 20). The term TGF-f3 actually describes a family of closely related polypeptide growth factors; three isoforms are found in mammals (TGF-f31-3; Refs. 21-23), a fourth (TGF-134) in the chicken =-=(24)-=-, and a fifth (TGF-f35) in Xenopus (25). All five proteins are produced as inactive complexes and are dissociated (by proteolysis, for example) to produce active molecules (26). The active molecules a...

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...it is this increase in the number of molecules of phosphorylated Cx43 per cell that is responsible for the increased GJC. In addition, these data indicate that Cx43 is not a substrate for the type II =-=(33)-=- and type V (34) receptors, both of which are serine/threonine kinases. The situation with regard to serine/threonine phosphorylation of Cx43 is complex; activators of protein kinase A have been shown...

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... that induced by TGF-31 (Fig. 5). Differences in the potencies of TGF- isoforms have been meported previously; j31 appears more active in endothelial cells (61), lL-3-stimulated hematopoietic cells =-=(62)-=-, and MCF-7 cells (63). In contrast, f32 is more potent in a Xenopus mesoderm induction assay (64) and the growth stimulation of fibroblasts (63). For a number of years, our laboratory has presented d...

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...have a functional role in tissue homeostasis, in nutrient and excretory product transfer in the avasculan lens and cornea (1 ), and in the transmission of momphogenic signals in embryonic development =-=(6)-=-. A role for gap junctions in growth control has also been proposed (1). Direct experimental support for this hypothesis comes from observations that growth of transformed cells can be inhibited by th...

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...ned in contrast to their only occasional presence in controls. These immunofluorescent images obtained after TGF-3treatment(Fig. 7)were qualitatively similarto images of retinoid treated lOT1h cells =-=(12)-=-. The similarity between plaque size and distribution in cells treated with TGF-i at 1 and 1 0 ng/ml, in spite of clearly increased GJC (Table 1) and total Cx43 (Fig. 2) at the higher TGF-f3 concentr...

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... type II receptors (38). The situation, however, is complex because the final cellular response to TGF-3 depends upon receptor availability as a result of interactions between receptor types I and II =-=(32, 37)-=-. Here we have tested the relationship between growth control and GJC by examining the influence of TGF-f3 on these parameters in iOTt/2 cells. The model ofgmowth control via GJC discussed above would...

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...ase in the number of molecules of phosphorylated Cx43 per cell that is responsible for the increased GJC. In addition, these data indicate that Cx43 is not a substrate for the type II (33) and type V =-=(34)-=- receptors, both of which are serine/threonine kinases. The situation with regard to serine/threonine phosphorylation of Cx43 is complex; activators of protein kinase A have been shown to increase Cx4...

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...inases (32-34). The type Ill receptor has also been cloned (35) and is a pnoteoglycan that acts to regulate the access of TGF-f3 to the type I and II signaling receptors (36). Receptor types I and II =-=(37)-=- mediate the effects of TGF-; type I mediates effects upon extracellular matrix proteins, while growth inhibition is mediated via type II receptors (38). The situation, however, is complex because th...

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...and are therefore considered to be unrelated to Cx43. A nonspecific band, co-migrating with Cx43 was also seen in the immunoprecipitates. This band has been reported by others and may represent actin =-=(39)-=-. In keeping with previously presented data (Fig. 2,A and B), total Cx43 protein levels were increased at least 3-fold by TGF-f31 and 2 (Fig. 6A). Increases in 32P-labeled Cx43 were also seen to resu...

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...ed epithelial cells, both in vitro (14, 15) and in vivo (40). In contrast, TGF-f3 is mitogenic for many fibmoblastic cells (16, 18) and can induce fibrosis after local (41) or systemic administration =-=(42)-=-. While details of the mechanisms ofTGF-j3 action remain unclear, the mitogenic effects are thought to be indirect, involving induction of platelet derived growth factor A and B chain (43). This labor...

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... ). Mitogenic compounds should therefore be expected to decrease GJC. Indeed, this prediction is satisfied by the association of serum-induced DNA synthesis in 3T3 fibroblasts with suppression of GJC =-=(44)-=-. Predictions of the effects of TGF-3 which, depending upon cell type, can act both as a mitogen and as an inhibitor of proliferation are not so straightforward. TGF-3 has been reported to both redu...

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...e effects of TGF-3 which, depending upon cell type, can act both as a mitogen and as an inhibitor of proliferation are not so straightforward. TGF-3 has been reported to both reduce (45) or enhance =-=(46)-=- intercellular communication in normal rat kidney cells. In BALB/c 3T3 fibmoblasts, TGF-f3 is reported either to increase (46) or have no effect (47) upon communication. These conflicting mesuIts may ...

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...3 has been reported to both reduce (45) or enhance (46) intercellular communication in normal rat kidney cells. In BALB/c 3T3 fibmoblasts, TGF-f3 is reported either to increase (46) or have no effect =-=(47)-=- upon communication. These conflicting mesuIts may be the result of differences in culture conditions and in the cell type employed. For example, in these previous reports, TGF-f3 was added either to ...

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...a (46, 47) or together with EGF into defined media (45). EGF (either added or contained in serum) is known to influence GJC (45), and TGF-f3 is known to up-regulate the expression of the EGF receptor =-=(48)-=-. Thus, the presence of other growth factors could contribute to the effects observed. Our experiments, which were carried out under defined conditions in the absence of exogenous growth factors, are ...

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... since previous studies have shown that while unphosphorylated Cx43 is transported to the plasma membrane (51), subsequent phosphorylation on as yet undefined residues appears to be necessary for GJC =-=(51, 52)-=-. However it should be noted that the amount of phosphorylated Cx43, in proportion to the amount of total cell protein, marginally increased after TGF-/3 treatment (Fig. 6C), and it seems likely that ...