Altered structure of the hybrid cell surface proteoglycan of mammary epithelial cells in response to transforming growth factor-beta

Transforming growth factor beta (TGF-beta) is a polypeptide growth factor that affects the accumulation of extracellular matrix by many cell types. We have examined the ability of mouse mammary epithelial (NMuMG) cells to respond to TGF-beta and assessed the effect of the growth factor on the expression of their cell surface heparan sulfate/chondroitin sulfate hybrid proteoglycan. NMuMG cells respond maximally to 3 ng/ml TGF-beta and the response is consistent with occupancy of the type III receptor. However, cells that are polarized, as shown by sequestration of the cell surface PG at their basolateral surfaces, must have the growth factor supplied to that site for maximal response. Immunological quantification of proteoglycan core protein on treated cells suggests that the cells have an unchanging number of this proteoglycan at their cell surface. Nonetheless, metabolic labeling with radiosulfate shows a approximately 2.5-fold increase in 35SO4-glycosaminoglycans in this proteoglycan fraction, defined either by its lipophilic, antigenic, or cell surface properties. Kinetic studies indicate that the enhanced radiolabeling is due to augmented synthesis, rather than slower degradation. Analysis of the glycosaminoglycan composition of the proteoglycan shows an increased amount of chondroitin sulfate, suggesting that the increased labeling per cell may be attributed to an augmented synthesis of chondroitin sulfate glycosaminoglycan on the core protein that also bears heparan sulfate, thus altering the proportions of these two glycosaminoglycans on this hybrid proteoglycan. We conclude that TGF-beta may affect NMuMG cell behavior by altering the structure and thus the activity of this proteoglycan.     

Distinct oligomeric states of SMAD proteins in the transforming growth factor-beta pathway

Protein interactions are critical for the function of SMADs as mediators of transforming growth factor-beta (TGF-beta) signals. TGF-beta receptor phosphorylation of SMAD2 or SMAD3 causes their association with SMAD4 and accumulation in the nucleus where the SMAD complex binds cofactors that determine the choice of target genes. We provide evidence that in the basal state, SMADs 2, 3, and 4 form separate, strikingly different complexes. SMAD2 is found mostly as monomer, whereas the closely related SMAD3 exists in multiple oligomeric states. This difference is due to a unique structural element in the MH1 domain of SMAD2 that inhibits protein-protein interactions in the basal state. In contrast to SMAD2 and SMAD3, SMAD4 in the basal state is found mostly as a homo-oligomer, most likely a trimer. Upon cell stimulation with TGF-beta, SMAD proteins become engaged in a multitude of complexes ranging in size from SMAD2-SMAD4 heterodimers to assemblies of >650 kDa. The latter display the highest DNA binding affinity for the TGF-beta-response elements of JUNB and collagen 7. These observations, all validated with endogenous SMAD proteins, modify previous models regarding the assembly and activity of SMAD complexes in the TGF-beta pathway.     

Oncostatin M and transforming growth factor-beta 1 induce post-translational modification and hyaluronan binding to CD44 in lung-derived epithelial tumor cells

CD44, a receptor for hyaluronan (HA), has been implicated in tumor growth and metastasis. Most CD44-positive cells fail to exhibit constitutive HA receptor function but CD44-mediated HA binding on hematopoetic cells can be induced by antibody cross-linking of the receptor and by physiologic stimuli, including cytokines. We now demonstrate that oncostatin M (OSM) and transforming growth factor-beta1, cytokines known to regulate the growth of tumor cells, stimulate HA binding in lung epithelial-derived tumor cells. In lung epithelial-derived tumor cells, cytokine-induced binding resulted from post-translational modification of the receptor. OSM-induced HA binding was associated with a reduction in N-linked carbohydrate content of CD44. In addition, OSM induced HA binding via a novel mechanism requiring sulfation of chondroitin sulfate chains linked to CD44. The mechanism underlying transforming growth factor-beta1 induced HA binding was distinct from the effects of OSM. The data presented indicate that modulation of the glycosylation and sulfation of CD44 by cytokines provides mechanisms for regulating cell adhesion during tumor growth and metastasis.     

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Possible involvement of transforming growth factor-beta 1 and transforming growth factor-beta receptor type II during luteinization in the marmoset ovary

The expression of transforming growth factor-beta 1 (TGF-beta 1), and transforming growth factor-beta receptor type II (T beta R-II), were evaluated in periovulatory marmoset ovaries. Histochemical methods were used, in particular double-labelling techniques, in order to correlate growth factor/receptor expression with proliferation (Ki 67), apoptosis (TUNEL method) and luteinization (3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)). The latter was used as a luteinization marker. Periovulatory ovaries are especially suited for studying all aspects since they typically consist of small non-luteinized follicles, large luteinizing follicles and corpora lutea accessoria (Clas), which have developed from large luteinizing follicles. TGF-beta 1 and T beta R-II expression was found in luteinizing theca cells of large periovulatory follicles and in all luteal cells of Clas. Non-luteinized theca cells, including those of small follicles were always devoid of any immunostaining. Granulosa cells of small follicles were immunopositive for T beta R-II. Large follicles with granulosa cell immunoreactivity of both antibodies coexisted with non-reactive follicles of comparable size. The highest activity of the luteal marker enzyme 3 beta-HSD was co-localized in the same cells that expressed TGF-beta 1 and T beta R-II. The double-labelling experiments revealed that TGF-beta 1 and T beta R-II expression is not correlated with proliferation or apoptosis of follicular cells. Our results indicate that TGF-beta 1 and T beta R-II participate in differentiation processes, i.e. luteinization, rather than proliferation. In particular, the dynamics of T beta R-II expression appear highly related to the process of luteinization.     

Abrogation of transforming growth factor-beta signaling by SMAD7 inhibits collagen gel contraction of human dermal fibroblasts

Human fibroproliferative disorders like hypertrophic scarring of the skin are characterized by increased contractility and excess extracellular matrix synthesis. A beneficial role of transforming growth factor (TGF)-beta in wound healing was proposed; however, chronic stimulation by this cytokine leads to fibrosis. In the present report, the intracellular TGF-beta signaling in fibroblasts derived from hypertrophic scars and normal skin was examined. In an attempt to intervene in profibrogenic TGF-beta functions, ectopic expression of Smad7 or dominant negative Smads3/4 completely inhibited contractility of scar-derived and normal fibroblasts after suspension in collagen gels. Both cell types displayed constitutive Smad2/3 phosphorylation and (CAGA)9-MLP-Luc activity with expression and phosphorylation of Smad3 being predominant in hypertrophic scar-derived fibroblasts. Down-regulation of intrinsic signaling with various TGF-beta antagonists, e.g. soluble TGF-beta receptor, latency-associated peptide, and anti-TGF-beta1 antibodies, confirms autocrine TGF-beta stimulation of both cell populations. Further, Smad7 expression inhibited alpha1 (I) collagen and alpha-smooth muscle actin expression. In summary, our data indicate that autocrine TGF-beta/Smad signaling is involved in contractility and matrix gene expression of fibroblasts from normal and hypertrophic scars. Smad7 inhibits these processes and may exert beneficial effects on excessive scar formation.     

Cell-type-specific localization of transforming growth factor-beta 2 and transforming growth factor-beta 1 in the hamster ovary: differential regulation by follicle-stimulating hormone and luteinizing hormone.

Cell-type-specific localization and gonadotropin regulation of transforming growth factor-beta 1 (TGF-beta 1) and transforming growth factor-beta 2 (TGF-beta 2) in the hamster ovary were evaluated immunohistochemically under three conditions: (1) during the estrous cycle (Day 1 = estrus; Day 4 = proestrus); (2) after the blockade of periovulatory gonadotropin surges by phenobarbital, and (3) after FSH and/or LH treatment of long-term hypophysectomized hamsters. Ovarian TGF-beta 1 activity was primarily localized in theca and interstitial cells. The activity increased moderately but significantly after the preovulatory LH surge and reached a peak at 0900 h, Day 2 h; oocytes showed considerable activity. TGF-beta 1 immunoreactivity subsequently fell to low levels in theca-interstitial cells through 0900 h, Day 4. Significant TGF-beta 2 immunoreactivity appeared after the surge, mainly in the granulosa cells of both preantral and antral follicles; a few interstitial cells surrounding preantral follicles showed discrete staining. TGF-beta 2 immunoreactivity in granulosa cells and in interstitial cells next to preantral follicles reached a peak at 0900 h, Day 1, and persisted up to 0900 h, Day 2; oocytes showed no staining. Phenobarbital treatment blocked the appearance of TGF-beta 1 and TGF-beta 2 immunoreactivities at 1600 h, Day 4; however, a rebound in immunoreactivities was observed with the onset of the surge after a 1-day delay. Replacement of LH to long-term hypophysectomized hamsters resulted in a marked increase in TGF-beta 1 immunoreactivity in the interstitial cells, but FSH, although it induced follicular development, did not influence ovarian TGF-beta 1 activity. Treatment with FSH, however, induced a massive increase in TGF-beta 2 immunoreactivity in the granulosa cells of newly developed antral and preantral follicles but not in the interstitial cells; LH, on the other hand, had no significant effect on TGF-beta 2 activity. Treatment with FSH and LH combined resulted in a dramatic increase in TGF-beta 2 immunoreactivity in granulosa and interstitial cells and in TGF-beta 1 in theca and interstitial cells comparable to their peak activity in intact animals. Western analyses substantiated the presence of TGF-beta 1 and TGF-beta 2 in the hamster ovary and the specificity of immunolocalization. These studies, therefore, provide critical evidence that TGF-beta 1 and TGF-beta 2 in the hamster ovary are expressed in specific cell types and that their expression is differentially regulated by LH and FSH, respectively.     

Inhibition of proliferation of cultured rat liver epithelial cells at specific cell cycle stages by transforming growth factor-beta

Proliferation of early-passage propagable cultured rat liver epithelial cells derived from normal adult rats is markedly inhibited by transforming growth factor-beta (TGF-beta). Inhibition, which is completely reversible, is effected at two distinct points of the cell cycle, the G1/S border and the G0 or early G1 phase. With increasing passages in culture, hepatic epithelial cells progressively become less sensitive to the inhibitory effect of TGF-beta.     

Transcriptional activation of collagenase-3 by transforming growth factor-beta1 is via MAPK and Smad pathways in human breast cancer cells

Transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen-activated protein kinase (MAPK) and/or Smad pathways are involved in TGF-beta1-stimulated collagenase-3 expression in MDA-MB231 cells. Biochemical blockade of extracellular regulated kinase-1/2 and p38 MAPK pathways partially abolished TGF-beta1-stimulated collagenase-3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF-beta1-response. These data indicate that TGF-beta1-induced MAPK and Smad pathways are involved in TGF-beta1-stimulated collagenase-3 expression in MDA-MB231 cells.     

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-beta

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.