Expression of transforming growth factor-beta1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function.

Thyroid growth and function are intricately regulated by both positive and negative factors. In the present study, we have investigated the expression of transforming growth factor-beta (TGF-beta) super-family members and their receptors in normal porcine thyroid follicle cells. In tissue sections of porcine thyroids, we observed an expression of TGF-beta1, activin A, and bone morphogenetic protein (BMP)-7 proteins. The staining was localized to the follicular epithelium. In affinity cross-linking experiments, TGF-beta1 was found to bind to heteromeric complexes of TGF-beta type I and type II receptors, and activin A bound most efficiently to heteromeric complexes of activin type IB and type II receptors. We were unable to detect any BMP receptors (BMPRs) in attempts to perform affinity cross-linking with BMP-7. However, expression of BMPR-IA and BMPR-II messenger RNA (mRNA) was detected by Northern blot analysis. Both TGF-beta1 and activin A, but not BMP-7, increased the phosphorylation of Smad2, induced nuclear translocation of Smad2, Smad3, and Smad4, and inhibited thyrocyte cell growth as well as TSH-stimulated cAMP response. TGF-beta1 was more potent, compared with activin A, to induce these cellular responses. Taken together, our findings indicate a role for several members of the TGF-beta family in regulation of thyroid growth and function.     

Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension

Mutations in the bone morphogenetic protein type II receptor gene (BMPR2) are the major genetic cause of familial pulmonary arterial hypertension (FPAH). Although smooth muscle cell proliferation contributes to the vascular remodeling observed in PAH, the role of BMPs in this process and the impact of BMPR2 mutation remains unclear. Studies involving normal human pulmonary artery smooth muscle cells (PASMCs) suggest site-specific responses to BMPs. Thus, BMP-4 inhibited proliferation of PASMCs isolated from proximal pulmonary arteries, but stimulated proliferation of PASMCs from peripheral arteries, and conferred protection from apoptosis. These differences were not caused by differential activation of BMP signaling pathways because exogenous BMP-4 led to phosphorylation of Smad1, p38(MAPK), and ERK1/2 in both cell types. However, the proproliferative effect of BMP-4 on peripheral PASMCs was found to be p38MAPK/ERK-dependent. Conversely, overexpression of dominant-negative Smad1 converted the response to BMP-4 in proximal PASMCs from inhibitory to proliferative. Furthermore, we confirmed that proximal PASMCs harboring kinase domain mutations in BMPR2 are deficient in Smad signaling and are unresponsive to the growth suppressive effect of BMP-4. Moreover, we show that the pulmonary vasculature of patients with familial and idiopathic PAH are deficient in the activated form of Smad1. We conclude that defective Smad signaling and unopposed p38(MAPK)/ERK signaling, as a consequence of mutation in BMPR2, underlie the abnormal vascular cell proliferation observed in familial PAH.     

Bone morphogenetic protein 2 is expressed by, and acts upon, mature epithelial cells in the colon

BACKGROUND AND AIMS: The recent findings of bone morphogenetic protein (BMP) receptor Ia mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer suggest a role for BMPs in the colonic epithelium and colon cancer. We investigated the role of BMP2 in the colon. METHODS: We assessed BMP receptor expression in cell lines using the reverse-transcribed polymerase chain reaction and immunoblotting. We investigated the effect of BMP2 on cell lines using the MTT assay and by immunoblotting for markers of differentiation, proliferation, and apoptosis. We assessed the expression of BMP2, its receptors, and signal transduction elements in mouse and human colon tissue using immunohistochemistry. We also investigated the effect of the BMP antagonist noggin in vivo in mice by assessing colon tissue with immunohistochemistry and immunoblotting. Finally, we investigated the expression of BMP2 in microadenomas from familial adenomatous polyposis patients. RESULTS: BMP receptors (BMPR) Ia, BMPR Ib, and BMPR II are all expressed in colonic epithelial cell lines. BMP2 inhibits colonic epithelial cell growth in vitro, promoting apoptosis and differentiation and inhibiting proliferation. BMP2, BMPRIa, BMPRIb, BMPRII, phosphorylated Smad1, and Smad4 are expressed predominantly in mature colonocytes at the epithelial surface in normal adult human and mouse colon. Noggin inhibits apoptosis and proliferation in mouse colonic epithelium in vivo. BMP2 expression is lost in the microadenomas of familial adenomatous polyposis patients. CONCLUSIONS: These data suggest that BMP2 acts as a tumor suppressor promoting apoptosis in mature colonic epithelial cells.     

IIIc isoform of fibroblast growth factor receptor 1 is overexpressed in human pancreatic cancer and enhances tumorigenicity of hamster ductal cells

BACKGROUND & AIMS: Fibroblast growth factors (FGFs) are mitogenic polypeptides that signal via FGF receptors (FGFRs). Pancreatic ductal adenocarcinomas (PDACs) overexpress multiple FGFs, implying a potential for growth modulation. In this study we investigated the importance of the IIIc splice variant of FGFR-1 (FGFR-1 IIIc) in PDAC. METHODS: Expression of FGFR-1 IIIc was determined by a ribonuclease protection assay in pancreatic cancer cell lines and in tissues. In situ hybridization was used to localize FGFR-1 IIIc messenger RNA (mRNA) in pancreatic tissues. A cDNA encoding FGFR-1 IIIc was stably transfected into the well-differentiated TAKA-1 pancreatic ductal cell line that is not responsive to FGF5 and does not express FGFR-1. RESULTS: FGFR-1 IIIc was expressed in 5 of 7 pancreatic cancer cell lines and in the majority of the cancer cells in 4 of 7 PDAC samples. In vitro, TAKA-1 cells stably transfected with FGFR-1 IIIc exhibited increased basal growth; enhanced basal tyrosine phosphorylation of FGFR substrate-2 (FRS2), Shc, and phospholipase Cgamma; and increased activation of mitogen-activated protein kinase (MAPK). PD98059, an inhibitor of MAPK, suppressed the basal growth of parental and transfected clones, but the effect was more marked in clones expressing FGFR-1 IIIc. In vivo, tumor formation in nude mice was dramatically enhanced with FGFR-1 IIIc transfected (20 of 20) in comparison with sham transfected (0 of 10) cells. CONCLUSIONS: Our data indicate that FGFR-1 IIIc is expressed in human pancreatic cancer cells, promotes mitogenic signaling via the FRS2-MAPK pathway, and has the potential to enhance pancreatic ductal cell transformation.     

Ligand induced upregulation of the type II transforming growth factor (TGF-beta) receptor enhances TGF-beta responsiveness in COLO-357 cells

The effects of transforming growth factor (TGF)-beta 1 on type I and type II TGF-beta receptor (T beta RI and T beta RII) expression were examined in five pancreatic cancer cell lines. In contrast to its actions in COLO-357, a TGF-beta-sensitive pancreatic cancer cell line, TGF-beta 1 did not significantly alter TGF-beta receptor expression in either the TGF-beta-sensitive BXPC-3 and PANC-1 cells or in the TGF-beta-resistant CAPAN-1 and T3M4 cells. Neutralizing anti-T beta RII antibodies blocked TGF-beta 1-dependent signaling in COLO-357 cells but exhibited an attenuated effect in COLO-357 cells preincubated with TGF-beta 1 for 48 h. Basal T beta RII expression levels were comparable in all five cell lines examined. In contrast, COLO-357 cells and BX-PC-3 cells expressed relatively high basal levels of T beta RI. However, COLO-357 cells harbored a normal Smad4 gene, whereas BX-PC-3 cells exhibited a complete deletion of this gene. We conclude that the TGF-beta 1-induced T beta RII upregulation serves to enhance TGF-beta 1 responsiveness in COLO-357 cells, and that this upregulation requires the presence of adequate levels of T beta RI and T beta RII, and a functional Smad4 gene product. Our findings also indicate that TGF-beta 1 may inhibit pancreatic cancer cell growth via a Smad4-independent pathway.     

Adenovirus-mediated transfer of a truncated fibroblast growth factor (FGF) type I receptor blocks FGF-2 signaling in multiple pancreatic cancer cell lines

Pancreatic ductal adenocarcinomas (PDACs) overexpress several members of the fibroblast growth factor (FGF) family of ligands and the type I FGF receptor (FGFR-1), and enhanced FGF-2 protein levels correlate with shorter postoperative survival of patients with PDAC. In this study, we investigated the effects of FGF-2 on cell proliferation and mitogen-activated protein kinase (MAPK) activation before and after abrogation of FGFR-1-dependent signaling in 4 pancreatic cancer cell lines (ASPC-1, COLO-357, MIA-PaCa-2, and PANC-1). Signaling was blocked by infecting the cells with an adenoviral vector encoding for a truncated FGFR-1 (AdtrFGFR-1). FGF-2 enhanced the growth of all 4 cell lines and activated MAPK in 3 of these cell lines. Infection with the AdtrFGFR-1 virus resulted in abundant expression of the truncated FGFR-1 at the RNA and protein level, markedly attenuated FGF-2-induced proliferation in all 4 tested cell lines, and decreased FGF-2-dependent MAPK activation in the 3 cell lines in which FGF-2 activated this pathway. These findings suggest that FGFR-1-mediated mitogenesis in multiple pancreatic cancer cells can be efficiently blocked with an adenoviral vector encoding a truncated FGFR-1, raising the possibility that AdtrFGFR-1 may ultimately have a therapeutic role in PDAC.     

Down-regulation of the dual-specificity phosphatase MKP-1 suppresses tumorigenicity of pancreatic cancer cells

BACKGROUND & AIMS: In both pancreatic cancer and chronic pancreatitis, there is enhanced expression of mitogenic growth factors and their tyrosine kinase receptors, which have the capacity to activate mitogen-activated protein kinase (MAPK). In view of the important role of MAPK kinase phosphatase (MKP)-1 in the regulation of MAPK activation, the expression and functional role of MKP-1 was analyzed. METHODS: Pancreatic tissues were analyzed by Northern blotting, Western blotting, and immunohistochemistry. Pancreatic cancer cells were transfected with a full-length MKP-1 antisense construct. Growth characteristics and tumorigenicity in vivo and the effects of mitogenic growth factors on cell growth and MAPK activation were determined in transfected and control cells. RESULTS: MKP-1 messenger RNA (mRNA) levels were increased in pancreatic cancer and chronic pancreatitis (CP) tissues. Moderate to strong MKP-1 immunoreactivity was present in the cancer cells, ductal cells of pancreatic intraepithelial neoplasia, and in tubular complexes in CP. Down-regulation of MKP-1 resulted in decreased anchorage-dependent and -independent growth of pancreatic cancer cells, and decreased tumorigenicity in a nude mouse tumor model. MKP-1 down-regulation led to decreased proliferation and sustained MAPK activation in response to mitogens. CONCLUSIONS: Suppression of MKP-1 expression reduces the tumorigenicity of pancreatic cancer cells in vivo, suggesting that MKP-1 contributes to enhanced mitogenic signaling in pancreatic cancer cells.     

Functional interaction of fibroblast growth factor-8, bone morphogenetic protein and estrogen receptor in breast cancer cell proliferation

Estrogen is involved in the development and progression of breast cancer. Here we investigated the effect of fibroblast growth factor (FGF)-8 on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptor (ER)α, ERβ, FGF receptors, and Smad signaling molecules. Estradiol stimulated MCF-7 cell proliferation in a concentration-responsive manner, whereas BSA-bound estradiol had a weak effect on MCF-7 cell mitosis compared with the effect of free estradiol. It is notable that estrogen-induced cell proliferation was enhanced in the presence of FGF-8 and that the combined effects were reversed in the presence of an FGF-receptor kinase inhibitor or an ER antagonist. It was also revealed that FGF-8 increased the expression levels of ERα, ERβ and aromatase mRNAs, while estradiol reduced the expression levels of ERs, aromatase and steroid sulfatase in MCF-7 cells. FGF-8-induced phosphorylation of FGF receptors was augmented by estradiol, which was reversed by an ER antagonist. FGF-8-induced activation of MAPKs and AKT signaling was also upregulated in the presence of estrogen. On the other hand, FGF-8 suppressed BMP-7 actions that are linked to mitotic inhibition by activating the cell cycle regulator cdc2. FGF-8 was revealed to inhibit BMP receptor actions including Id-1 promoter activity and Smad1/5/8 phosphorylation by suppressing expression of BMP type-II receptors and by increasing expression of inhibitory Smads. Collectively, the results indicate that FGF-8 acts to facilitate cell proliferation by upregulating endogenous estrogenic actions as well as by suppressing BMP receptor signaling in ER-expressing breast cancer cells.     

Involvement of bone morphogenetic protein 4 (BMP-4) in pituitary prolactinoma pathogenesis through a Smad/estrogen receptor crosstalk

Pituitary tumor development involves clonal expansion stimulated by hormones and growth factorscytokines. Using mRNA differential display, we found that the bone morphogenetic protein (BMP) inhibitor noggin is down-regulated in prolactinomas from dopamine D2-receptor-deficient mice. BMP-4 is overexpressed in prolactinomas taken from dopamine D2-receptor-deficient female mice, but expression of the highly homologous BMP-2 does not differ in normal pituitary tissue and prolactinomas. BMP-4 is overexpressed in other prolactinoma models, including estradiol-induced rat prolactinomas and human prolactinomas, compared with normal tissue and other pituitary adenoma types (Western blot analysis of 48 tumors). BMP-4 stimulates, and noggin blocks, cell proliferation and the expression of c-Myc in human prolactinomas, whereas BMP-4 has no action in other human pituitary tumors. GH3 cells stably transfected with a dominant negative of Smad4 (Smad4dn; a BMP signal cotransducer) or noggin have reduced tumorigenicity in nude mice. Tumor growth recovered in vivo when the Smad4dn expression was lost, proving that BMP-4Smad4 are involved in tumor development in vivo. BMP-4 and estrogens act through overlapping intracellular signaling mechanisms on GH3 cell proliferation and c-myc expression: they had additive effects at low concentrations but not at saturating doses, and their action was inhibited by blocking either pathway with the reciprocal antagonist (i.e., BMP-4 with ICI 182780 or 17beta-estradiol with Smad4dn). Furthermore, coimmunoprecipitation studies demonstrate that under BMP-4 stimulation Smad4 and Smad1 physically interact with the estrogen receptor. This previously undescribed prolactinoma pathogenesis mechanism may participate in tumorigenicity in other cells where estrogens and the type beta transforming growth factor family have important roles.     

Expression of the IIIc variant of FGF receptor-1 confers mitogenic responsiveness to heparin and FGF-5 in TAKA-1 pancreatic ductal cells.

BACKGROUND: Fibroblast growth factors (FGFs) contribute to angiogenesis and mitogenesis by binding to tyrosine kinase receptors termed FGF receptors (FGFRs). FGF-5 is a secreted FGF that is believed to preferentially act via the IIIc splice variant of FGFR-1. Human pancreatic ductal carcinoma cells express FGF-5 and FGFR-1IIIc, implying a potential for autocrine growth modulation. AIM: In this study we investigated the importance of FGFR-1 IIIc expression for FGF-5 mitogenic signaling in a pancreatic ductal cell line. METHODS: A cDNA encoding FGFR-1 IIIc was expressed in the well-differentiated TAKA-1 Syrian hamster pancreatic ductal cell line. RESULTS: TAKA-1 cells secrete FGF-5, but were found not to express FGFR-1 and to be unresponsive to exogenous FGF-5. In contrast, TAKA-1 clones expressing FGFR-1 IIIc were growth stimulated in the presence of FGF-5 and displayed enhanced mitogen-activated protein kinase (MAPK) activity in the presence of FGF-5. PD98059, an inhibitor of this pathway, inhibited FGF-5-induced growth in these clones. CONCLUSION: Our data demonstrate that FGFR-1 IIIc can mediate FGF-5-induced mitogenesis via the MAPK pathway in pancreatic ductal cells, and suggest that expression of FGFR-1 IIIc in conjunction with FGF-5 may contribute to the pathobiology of human pancreatic cancer.     

Bone morphogenetic proteins (BMP) -4, -6, and -7 potently suppress basal and luteinizing hormone-induced androgen production by bovine theca interna cells in primary culture: could ovarian hyperandrogenic dysfunction be caused by a defect in thecal BMP signaling?

We reported recently that bovine theca interna cells in primary culture express several type-I and type-II receptors for bone morphogenetic proteins (BMPs). The same cells express at least two potential ligands for these receptors (BMP-4 and -7), whereas bovine granulosa cells and oocytes express BMP-6. Therefore, BMPs of intrafollicular origin may exert autocrine/paracrine actions to modulate theca cell function. Here we report that BMP-4, -6, and -7 potently suppress both basal (P < 0.0001; respective IC(50) values, 0.78, 0.30, and 1.50 ng/ml) and LH-induced (P < 0.0001; respective IC(50) values, 5.00, 0.55, and 4.55 ng/ml) androgen production by bovine theca cells while having only a moderate effect on progesterone production and cell number. Semiquantitative RT-PCR showed that all three BMPs markedly reduced steady-state levels of mRNA for P450c17. Levels of mRNA encoding steroidogenic acute regulatory protein, P450scc, and 3beta-hydroxy- steroid dehydrogenase were also reduced but to a much lesser extent. Immunocytochemistry confirmed a marked reduction in cellular content of P450c17 protein after BMP treatment (P < 0.001). Exposure to BMPs led to cellular accumulation of phosphorylated Smad1, but not Smad2, confirming that the receptors signal via a Smad1 pathway. The specificity of the BMP response was further explored by coincubating cells with BMPs and several potential BMP antagonists, chordin, gremlin, and follistatin. Gremlin and chordin were found to be effective antagonists of BMP-4 and -7, respectively, and the observation that both antagonists enhanced (P < 0.01) androgen production in the absence of exogenous BMP suggests an autocrine/paracrine role for theca-derived BMP-4 and -7 in modulating androgen production. Collectively, these data indicate that an intrafollicular BMP signaling pathway contributes to the negative regulation of thecal androgen production and that ovarian hyperandrogenic dysfunction could be a result of a defective autoregulatory pathway involving thecal BMP signaling.     

Expression of the IIIc Variant of FGF Receptor-1 Confers Mitogenic Responsiveness to Heparin and FGF-5 in TAKA-1 Pancreatic Ductal Cells

Summary Background. Fibroblast growth factors (FGFs) contribute to angiogenesis and mitogenesis by binding to tyrosine kinase receptors termed FGF receptors (FGFRs). FGF-5 is a secreted FGF that is believed to preferentially act via the IIIc splice variant of FGFR-1. Human pancreatic ductal carcinoma cells express FGF-5 and FGFR-1 IIIc, implying a potential for autocrine growth modulation. Aim. In this study we investigated the importance of FGFR-1 IIIc expression for FGF-5 mitogenic signaling in a pancreatic ductal cell line. Methods. A cDNA encoding FGFR-1 IIIc was expressed in the well-differentiated TAKA-1 Syrian hamster pancreatic ductal cell line. Results. TAKA-1 cells secrete FGF-5, but were found not to express FGFR-1 and to be unresponsive to exogenous FGF-5. In contrast, TAKA-1 clones expressing FGFR-1 IIIc were growth stimulated in the presence of FGF-5 and displayed enhanced mitogen-activated protein kinase (MAPK) activity in the presence of FGF-5. PD98059, an inhibitor of this pathway, inhibited FGF-5-induced growth in these clones. Conclusion. Our data demonstrate that FGFR-1 IIIc can mediate FGF-5-induced mitogenesis via the MAPK pathway in pancreatic ductal cells, and suggest that expression of FGFR-1 IIIc in conjunction with FGF-5 may contribute to the pathobiology of human pancreatic cancer.     

Interleukin-13 exerts autocrine growth-promoting effects on human pancreatic cancer, and its expression correlates with a propensity for lymph node metastases

Background and aims  Interleukin-13 (IL-13) is an anti-inflammatory cytokine produced in cells of hematopoetic origin. It is not known whether pancreatic cancer cells produce IL-13 or whether IL-13 can modulate pancreatic cancer cell growth and influence the frequency of lymph node metastases. Materials and methods  Cell growth and signaling were analyzed by cell counting, colorimetric proliferation assays, fluorescent-activated cell sorting, and in vitro kinase activity assays. IL-13 expression and secretion were determined by Northern blot analysis and enzyme-linked immunosorbent assay, respectively. Localization of IL-13 and its transmembrane receptor (IL-4R) in primary pancreatic ductal adenocarcinoma (PDAC) was characterized by immunohistochemistry. Results  IL-13 enhanced the growth of ASPC-1, CAPAN-1, and COLO-357 cells. This was associated with enhanced p44/42 mitogen-activated protein kinase (MAPK) phoshorylation. In contrast to p44/42 MAPK, phosphatidylinositol 3-kinase activity was also induced in IL-13-unresponsive MIA PaCa-2, PANC-1, and T3M4 cells. All cells expressed and secreted IL-13. Neutralizing IL-13 antibodies inhibited the growth of ASPC-1 and CAPAN-1 cells. Immunohistochemical analysis of resected primary ductal adenocarcinoma specimens revealed high levels of IL-13 in 30 of 70 cases and its transmembrane receptor (IL-4R) in 28 of 70 cases, respectively. Fifteen of 16 specimens (94%) exhibiting high IL-13 and IL-4R coexpression had lymph node metastases, while only 30 of the remaining 54 samples (56%) had positive lymph nodes (p = 0.0134). Conclusion  IL-13 can act as an autocrine growth factor in PDAC. Endogenous expression of IL-13 in conjunction with IL-4R in the cancer cells seems to facilitate lymph node metastasis. Andrea Formentini and Olga Prokopchuk have equally contributed to this work.     

Crosstalk between tyrosine kinase receptors,GSK3 and BMP2 signaling during osteoblastic differentiation of human mesenchymal stem cells

Bone morphogenic proteins (BMPs) promote mesenchymal stem cell (MSC) osteogenic differentiation, whereas platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) activate their proliferation through receptors tyrosine kinase (RTK). The effects of PDGF or FGF receptor signaling pathway on BMP2-induced osteoblastic differentiation was investigated in human MSC (HMSC). Inhibition of PDGF or/and FGF receptors enhanced BMP2-induced alkaline phosphatase (ALP) activity, expression of Osterix, ALP and Bone sialoprotein, and matrix calcification. These effects were associated with increased Smad-1 activity, indicating that mitogenic factors interfere with Smad signaling in HMSC differentiation. RTK activate MAPK and inhibit GSK3 through the PI3K/Akt pathway. Biochemical analysis indicated that MAPK JNK and GSK3 especially are potential signaling molecules regulating BMP-induced osteoblastic HMSC differentiation. These observations highlight that the osteogenic effects of BMP2 are modulated by mitogenic factors acting through RTK.