Complexity in interpretation of embryonic epithelial-mesenchymal transition in response to transforming growth factor-beta signaling

Epithelial-mesenchymal transition (EMT) is a highly conserved and fundamental process that governs morphogenesis in development and may also contribute to cancer metastasis. Transforming growth factor (TGF-beta) is a potent inducer of EMT in various developmental and tumor systems. The analysis of TGF-beta signal transduction pathways is now considered a critically important area of biology, since many defects occur in these pathways in embryonic development. The complexity of TGF-beta signal transduction networks is overwhelming due to the large numbers of interacting constituents, complicated feedforward, feedback and crosstalk circuitry mechanisms that they involve in addition to the cellular kinetics and enzymatics that contribute to cell signaling. As a result of this complexity, apparently simple but highly important questions remain unanswered, that is, how do epithelial cells respond to such TGF-beta signals? System biology and cellular kinetics play a crucial role in cellular function; omissions of such a critical contributor may lead to inaccurate understanding of embryonic EMT. In this review, we identify and explain why certain conditions need to be considered for a true representation of TGF-beta signaling in vivo to better understand the controlled, yet delicate mechanism of embryonic EMT.     

Multiple transforming growth factor-beta isoforms and receptors function during epithelial-mesenchymal cell transformation in the embryonic heart

Epithelial-mesenchymal cell transformation (EMT) is a critical process during development of the heart valves. Transition of endothelial cells into mesenchymal cells in the atrioventricular (AV) canal and the outflow tract regions of the heart form the cardiac cushions that eventually form the heart valves. Collagen gel invasion assay has aided in the identification of molecules that regulate EMT. Among those, transforming growth factor-beta (TGF-beta) ligands and receptors demonstrate a critical role during EMT. In the chick, TGF-beta ligands and some receptors have specific functions during EMT. TGF-beta2 mediates endothelial cell-cell activation and separation, and TGF-beta3 mediates cell invasion into the extracellular matrix. Receptors involved in the EMT process include TGF-beta receptor type II (TBRII), TBRIII, endoglin and the TBRI receptors, ALK2 and ALK5. In contrast, in the mouse model, TGF-beta2 is the only ligand involved in EMT. The TGF-beta2 null mouse has either increased EMT or a mesenchymal cell proliferation after EMT. However, functional studies of TGF-beta1 in vivo and in vitro showed that TGF-beta1 functions in the EMT of the mouse AV canal. Latent TGF-beta-binding protein (LTBP-1) and endoglin have a role in the EMT process. Therefore, TGF-betas mediate cardiac EMT in both embryonic species. Further studies will reveal the identification of ligand and receptor-specific activities.     

Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis.

Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis.     

Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) can regulate cell growth and differentiation as well as production of extracellular matrix proteins. Elevated production of TGF-beta has been associated with human and rodent chronic inflammatory and fibrotic diseases. Using immunohistochemical staining, we have examined lung sections of patients with advanced idiopathic pulmonary fibrosis (IPF), a disease characterized by chronic inflammation and fibrosis and demonstrated a marked and consistent increase in TGF-beta production in epithelial cells and macrophages when compared to patients with nonspecific inflammation and those with no inflammation or fibrosis. In patients with advanced IPF, intracellular staining with anti-LC (1-30) TGF-beta antibody was seen prominently in bronchiolar epithelial cells. In addition, epithelial cells of honeycomb cysts and hyperplastic type II pneumocytes stained intensely. Anti-CC (1-30) TGF-beta antibody, which reacts with extracellular TGF-beta, was localized in the lamina propria of bronchioles and in subepithelial regions of honeycomb cysts in areas of dense fibroconnective tissue deposition. The close association of subepithelial TGF-beta to the intracellular form in advanced IPF suggests that TGF-beta was produced and secreted primarily by epithelial cells. Because of the well-known effects of TGF-beta on extracellular matrix formation and on epithelial cell differentiation, the increased production of TGF-beta in advanced IPF may be pathogenic to the pulmonary fibrotic and regenerative responses seen in this disease.     

Regulation of extracellular matrix remodeling following transforming growth factor-beta1/epidermal growth factor-stimulated epithelial-mesenchymal transition in human premalignant keratinocytes

During tumor progression, malignant cells exploit critical developmental and tissue remodeling programs, often promoting a plastic phenotype referred to as an epithelial-mesenchymal transition (EMT). Autocrine/paracrine signaling due to tumor microenvironment cytokines, such as members of the transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) families, largely regulates the morphological and invasive phases of the EMT phenotype. Notably, epithelial cell initiation often coincides with a switch in the response of these cells to TGF-beta and is concomitant with EGF receptor amplification. Modeling these events, we have observed that premalignant human keratinocytes, HaCaTs, acquire a highly motile and scattered phenotype indicative of EMT following stimulation with TGF-beta1 and EGF. TGF-beta1 and EGF have been shown to upregulate a number of matrix metalloproteinases (MMP) in epithelial cells, which may in turn play a role in developing metastatic potential in these cells. We have established that an increase in MMP-10 expression occurs following treatment of HaCaT cells with a combination of TGF-beta1 and EGF. This increase in MMP-10 expression paralleled the development of a collagenolytic phenotype that was sensitive to components of the plasminogen activation system, including the plasminogen activator inhibitor type-1 (PAI-1). Significantly high levels of MMP-10 have been detected in squamous cell carcinomas of the head and neck, esophagus, oral cavity and skin. Importantly, TGF-beta1 in addition to upregulating MMP-10 has been shown to upregulate PAI-1 expression in HaCaT cells. Taken together, these observations suggest that TGF-beta1 and EGF play a complex role in modulating proteolytic and transitional events such as EMT that may facilitate the progression of human premalignant epithelial cells toward a more invasive phenotype.     

Transforming growth factor-beta stimulates epithelial-mesenchymal transformation in the proepicardium.

The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE-derived cells undergoes epithelial-mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor-beta (TGFbeta) 1 and TGFbeta2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFbeta receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE-derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFbeta stimulates transformation in the PE and suggest that ALK2 partially mediates this effect.     

Expression of transforming growth factor-beta1 and transforming growth factor-beta receptors in hepatocellular carcinoma and dysplastic nodules.

In this study we analyzed by immunohistochemistry the expression of TGF-beta1 protein and TGF-beta receptors I and II in 4 low-grade dysplastic nodules, 2 high-grade dysplastic nodules, 6 early, 22 small, and 62 advanced hepatocellular carcinomas. The expression of TGF-beta1 protein by hepatocytes was decreased in advanced hepatocellular carcinoma compared with small or early hepatocellular carcinoma(P < .05). Frequent and intense staining of TGF-beta1 protein was noted in the sinusoidal endothelium of advanced hepatocellular carcinomas despite of its decreased staining in hepatocellular carcinoma cells. Reduced expression of TGF-beta receptors I and II compared with surrounding nontumorous tissue were noted from the early hepatocellular carcinoma stage suggesting that down-regulation of TGF-beta receptors is correlated with progression from premalignant to malignant phenotype. Reduced expression of both TGF-beta1 and TGF-beta receptor II in neoplastic hepatocytes were also significantly correlated with increased tumor size and increased proliferative activity(P < .05). These findings suggest that during hepatocarcinogenesis, the inhibitory effects of TGF-beta1 protein on hepatocellular carcinoma cells is outweighed by its effects on stromal elements, which, overall, contributes indirectly to a tumor growth stimulatory environment. Also, the growth-inhibitory effects of TGF-beta1 may have been further negated by reduced TGF-beta receptors on hepatocellular carcinoma cells.     

Expression of the type-1 repeats of thrombospondin-1 inhibits tumor growth through activation of transforming growth factor-beta

In the present study, the type-1 repeats of thrombospondin-1 (TSP-1) were transfected into A431 cells. Expression of all three type-1 repeats (3TSR) and expression of just the second type-1 repeat containing the transforming growth factor (TGF)-beta activating sequence KRFK (TSR2 + KRFK) significantly inhibited in vivo tumor angiogenesis and growth in nude mice. These tumors expressed increased levels of both active and total TGF-beta. A431 cells expressing the second type-1 repeat without the KRFK sequence (TSR2 - KRFK) produced tumors that were slightly larger than the 3TSR and TSR2 + KRFK tumors. These tumors expressed elevated levels of active TGF-beta but levels of total TGF-beta were not different from control tumors. Injection of the peptide, LSKL, which blocks TSP-1 activation of TGF-beta, reversed the growth inhibition observed with cells expressing TSR2 + KRFK to a level comparable to controls. Various residues in the WSHWSPW region and the VTCG sequence of both TSR2+/- KRFK were mutated. Although mutation of the VTCG sequence had no significant effect on tumor growth, mutation of the WSHWSPW sequence reduced inhibition of tumor growth. These findings suggest that the inhibition of tumor angiogenesis and growth by endogenous TSP-1 involves regulation of both active and total TGF-beta and the sequences KRFK and WSHWSPW in the second type-1 repeat.     

The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts

Wound contraction is mediated by myofibroblasts, specialized fibroblasts that appear in large numbers as the wound matures and when resistance to contractile forces increases. We considered that the regulation of myofibroblast differentiation by wound-healing cytokines may be dependent on the resistance of the connective tissue matrix to deformation. We examined transforming growth factor-beta1 (TGF-beta1) induction of the putative fibroblast contractile marker, alpha-smooth muscle actin (alpha-SMA), and the regulation of this process by the compliance of collagen substrates. Cells were cultured in three different types of collagen gels with wide variations of mechanical compliance as assessed by deformation testing. The resistance to collagen gel deformation determined the levels of intracellular tension as shown by staining for actin stress fibers. For cells plated on thin films of collagen-coated plastic (ie, minimal compliance and maximal intracellular tension), TGF-beta1 (10 ng/ml; 6 days) increased alpha-SMA protein content by ninefold as detected by Western blots but did not affect beta-actin content. Western blots of cells in anchored collagen gels (moderate compliance and tension) also showed a TGF-beta1-induced increase of alpha-SMA content, but the effect was greatly reduced compared with collagen-coated plastic (<3-fold increase). In floating collagen gels (high compliance and low tension), there were only minimal differences of alpha-SMA protein. Northern analyses for alpha-SMA and beta-actin indicated that TGF-beta1 selectively increased mRNA for alpha-SMA similar to the reported protein levels. In pulse-chase experiments, [35S]methionine-labeled intracellular alpha-SMA decayed most rapidly in floating gels, less rapidly in anchored gels, and not at all in collagen plates after TGF-beta1 treatment. TGF-beta1 increased alpha2 and beta1 integrin content by 50% in cells on collagen plates, but the increase was less marked on anchored gels and was undetectable in floating gels. When intracellular tension on collagen substrates was reduced by preincubating cells with blocking antibodies to the alpha2 and beta1 integrin subunits, TGF-beta1 failed to increase alpha-SMA protein content in all three types of collagen matrices. These data indicate that TGF-beta1-induced increases of alpha-SMA content are dependent on the resistance of the substrate to deformation and that the generation of intracellular tension is a central determinant of contractile cytoskeletal gene expression.     

Vascular remodeling in primary pulmonary hypertension. Potential role for transforming growth factor-beta.

Active exogenous transforming growth factor-beta s (TGF-beta s) are potent modulators of extracellular matrix synthesis in cell culture and stimulate matrix synthesis in wounds and other remodeling tissues. The role of endogenous TGF-beta s in remodeling tissues is less well defined. Vascular remodeling in the pulmonary arteries of patients with primary pulmonary hypertension is characterized, in part, by abnormal deposition of immunohistochemically detectable procollagen, thereby identifying actively remodeling vessels. We used this marker of active matrix synthesis to begin defining the in vivo role of TGF-beta in the complex milieu of actively remodeling tissues. Immunohistochemistry using isoform-specific anti-TGF-beta antibodies was performed to determine whether TGF-beta was present in actively remodeling hypertensive pulmonary arteries 20 to 500 microns in diameter. Intense, cell-associated TGF-beta 3 immunoreactivity was observed in the media and neointima of these hypertensive muscular arteries. Immunostaining was present, but less intense, in normal arteries of comparable size. TGF-beta 2 immunoreactivity was observed in normal vessels and was increased slightly in hypertensive vessels, in a pattern resembling TGF-beta 3 immunoreactivity. No staining was associated with the adventitia. TGF-beta 1 immunostaining was either faint or absent in both normal and hypertensive vessels. Comparison of procollagen and TGF-beta localization demonstrated that TGF-beta 2 and TGF-beta 3 colocalized at all sites of procollagen synthesis. However, TGF-beta was observed in vessels, or vascular compartments, where there was no procollagen synthesis. Procollagen immunoreactivity was not present in normal vessels that showed immunoreactivity for TGF-beta 2 and TGF-beta 3. These observations suggest: a) the stimulation of procollagen synthesis by TGF-beta in vivo is more complex than suggested by in vitro studies and b) a potential role for TGF-beta 2 or TGF-beta 3, but not TGF-beta 1, in hypertensive pulmonary vascular remodeling.     

Endogenous transforming growth factor-beta inhibits toll-like receptor mediated activation of human uterine natural killer cells

PROBLEM: Toll-like receptors (TLRs) recognition is an important means for the innate immune system to rapidly respond to pathogen invasion. Our aim was to determine whether uterine natural killer (uNK) cell cytokine production induced by stimulation through TLRs could be regulated by endogenous transforming growth factor (TGF)-beta in human endometrium. METHOD OF STUDY: Single cells were isolated from human endometrium, and interferon (IFN)-gamma production by endometrium cells and uNK cells was determined after stimulation by TLR agonists. The role of TGF-beta in regulating this response was tested by blocking TGF-beta function using antibodies or a specific inhibitor, SB431542. RESULTS: TGF-beta blockade increased TLR agonist induced IFN-gamma by uNK cells. The regulation of uNK cell cytokine production was observed when uNK cells were incubated with agonists for TLR2 (PGN) or TLR3 (polyI:C). Blockade of TGF-beta or TGF-beta receptor signaling had no effect on constitutive cytokine production in the absence of TLR agonists. CONCLUSION: The results indicate that endogenous TGF-beta alters cytokine responses of uNK cells in human endometrium in response to TLR agonists. These data suggest that uNK cell responses to microbial pathogens in the endometrium are regulated by the amount of biologically active TGF-beta present within the human endometrium.     

Melanoma-associated expression of transforming growth factor-beta isoforms.

Melanocytic neoplasia is characterized by the aberrant overproduction of multiple cytokines in vitro. However, it is largely unknown how cytokine expression relates to melanoma progression in vivo. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine commonly produced by cultured melanoma cells. The in situ expression of all three TGF-beta isoforms (TGF-beta1, -2, and -3) was determined immunohistochemically in melanocytes and in 51 melanocytic lesions using isoform-specific antibodies. Significant linear trends of expression were observed from melanocytes through nevi and primary and metastatic melanomas for all three isoforms. TGF-beta1 was expressed by some melanocytes and almost uniformly by nevi and melanomas. TGF-beta2 and -3 were not expressed in normal melanocytes but were expressed in nevi and early and advanced primary (radial and vertical growth phase) and metastatic melanomas in a tumor-progression-related manner. TGF-beta2 was heterogeneously expressed in advanced primary and metastatic melanomas, whereas TGF-beta3 was uniformly and highly expressed in these lesions. Thus, expression of TGF-beta isoforms in melanocytes and melanocytic lesions is heterogeneous and related to tumor progression, and expression of TGF-beta2 and TGF-beta3 commences at critical junctures during progression of nevi to primary melanomas.     

Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta

Tumor necrosis factor-alpha (TNF-alpha) is thought to be important in the development of pulmonary fibrosis. However, surfactant protein-C/TNF-alpha transgenic mice do not spontaneously develop pulmonary fibrosis but instead develop alveolar enlargement and loss of elastic recoil. We hypothesized that overexpression of TNF-alpha in the lung requires an additional insult to produce fibrosis. In this study we evaluated whether TNF-alpha overexpression altered the development of pulmonary fibrosis due to bleomycin or transforming growth factor-beta (TGF-beta). Either 0.2 U bleomycin or saline was administered into left lung of TNF-alpha transgenic mice and their transgene-negative littermates. To overexpress TGF-beta, an adenovirus vector containing either active TGF-beta (AdTGF-beta) or LacZ was administered at a dose of 3 x 108 plaque-forming units per mouse. Fibrosis was assessed histologically and by measurement of hydroxyproline. TNF-alpha transgenic mice tolerated bleomycin or AdTGF-beta, whereas the transgene-negative littermates demonstrated severe pulmonary fibrosis after either agent. An increase in prostaglandin E2 and downregulation of TNF receptor I expression were observed in the TNF-alpha transgenic mice. In addition, recombinant human TNF-alpha attenuated bleomycin-induced pulmonary fibrosis. TNF-alpha has a complex role in the development of pulmonary fibrosis. Endogenous TNF-alpha may be important in the development of fibrosis as indicated in other reports, but overexpression of TNF-alpha or exogenous TNF-alpha limits pulmonary fibrosis in mice.     

Morphine-induced macrophage apoptosis: the role of transforming growth factor-beta

Laboratory and clinical reports indicate that opiate addicts are prone to infections. This effect of opiates is partly attributed to opiate-induced macrophage (Mphi) apoptosis. In the present study, we evaluated the role of transforming growth factor-beta (TGF-beta) in morphine-induced apoptosis of murine J774 cells and peritoneal Mphi. Mphi harvested from morphine-treated mice showed greater (P < 0. 0001) apoptosis when compared with control Mphi. Morphine also enhanced apoptosis of J774 cells and peritoneal Mphi. Anti-TGF-beta antibody inhibited (P < 0.001) the morphine-induced apoptosis in J774 cells (control 0.7 +/- 0.4%; 10-6 M morphine 23.5 +/- 0.7%; anti-TGF-beta antibody (Ab) + 10-6 M morphine 8.1 +/- 0.7%; apoptotic cells/field) and peritoneal Mphi (control 1.5 +/- 0.9%; 10-6 M morphine 29.1 +/- 1.4%; 10-6 M morphine + anti-TGF-beta Ab 19. 1 +/- 1.8%; apoptotic cells/field). TGF-beta enhanced (P < 0.001) apoptosis of J774 cells and peritoneal Mphi. TGF-beta also promoted Mphi DNA fragmentation into integer multiples of 180 bp (ladder pattern). Immunocytochemical studies revealed that morphine enhanced the Mphi cytoplasmic content of TGF-beta. In addition, Western blotting showed increased production of TGF-beta by morphine-treated J774 cells when compared with control cells. Morphine increased J774 cell expression of bax. Interestingly, morphine-induced bax expression was inhibited by anti-TGF-beta Ab. As both morphine-induced J774 cell apoptosis and bax expression were inhibited by anti-TGF-beta Ab, it appears that morphine-induced J774 cell apoptosis may be mediated through the generation of TGF-beta.     

Abnormalities of the transforming growth factor-beta pathway in ocular melanoma

The majority of ocular melanomas occur in the uveal tract. Chemotherapy is generally ineffective and large tumours requiring enucleation have a greater than 50% mortality at 5 years. Monosomy for chromosome 3 is common in uveal melanoma and it is known that there is loss of responsiveness to transforming growth factor beta (TGFbeta) in melanoma cell lines. Since the gene for TGFbeta receptor II (TGFbetaR2) is located on chromosome 3p22, this study investigates the possibility that the TGFbeta pathway, and TGFbetaR2 in particular, might be involved in the pathogenesis of this rare eye tumour. To this end, the expression of molecules in the pathway has been examined by immunocytochemistry (TGFbeta, TGFbetaR2, SMAD2, SMAD3, SMAD4, and p27), backed up by a cell culture assay of TGFbeta-mediated growth suppression, RT-PCR for SMAD4, and loss of heterozygosity (LOH) on 3p22. There was LOH at 3p22 in 6/19 tumours and loss of TGFbetaR2 expression in 10/27 tumours. Immunohistochemistry for SMADs 2, 3, and 4 showed potential loss of signal transduction in 14/27 tumours. The results indicate abnormality of the TGFbeta pathway in 61% of tumours for which unequivocal results were obtained and suggest that abrogation of control of melanocyte growth by the TGFbeta pathway may be important in the formation of uveal melanoma.