Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor-α

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor-α (TGF-α) on these proteins. We observe that TGF-α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one-dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF-α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF-α in mouse preimplantation development. © 1993 Wiley-Liss, Inc.     

Modulation of gene expression in the preimplantation mouse embryo by TGF-α and TGF-β

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-α or TGF-β results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-α or TGF-β results in the increased expression of the b subunit of the F₀ ATPase. TGF-β also stimulates the expression of the DNA polymerase α. TGF-α treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin. © 1995 Wiley-Liss, Inc.     

Effects of transforming growth factor-β1 and vascular endothelial growth factor 165 gene transfer on Achilles tendon healing

Repaired Achilles tendons typically take weeks before they are strong enough to handle physiological loads. Gene therapy is a promising treatment for Achilles tendon defects. The aim of the present study was to evaluate the histological/biomechanical effects of Transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor 165 (VEGF₁₆₅) gene transfer on Achilles tendon healing in rabbits. Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) were transduced with adenovirus carrying human TGF-β1 cDNA (Ad-TGF-β1), human VEGF₁₆₅ cDNA (Ad-VEGF₁₆₅), or both (PIRES-TGF-β1/VEGF₁₆₅) Viruses, no cDNA (Ad-GFP), and the BMSCs without gene transfer and the intact tendon were used as control. BMSCs were surgically implanted into the experimentally injured Achilles tendons. TGF-β1 distribution, cellularity, nuclear aspect ratio, nuclear orientation angle, vascular number, collagen synthesis, and biomechanical features were measured at 1, 2, 4, and 8 weeks after surgery. The TGF-β1 and TGFβ1/VEGF₁₆₅ co-expression groups exhibited improved parameters compared with other groups, while the VEGF₁₆₅ expression group had a negative impact. In the co-expression group, the angiogenesis effects of VEGF₁₆₅ were diminished by TGF-β1, while the collagen synthesis effects of TGF-β1 were unaltered by VEGF₁₆₅. Thus treatment with TGF-β1 cDNA-transduced BMSCs grafts is a promising therapy for acceleration and improvement of tendon healing, leading to quicker recovery and improved biomechanical properties of Achilles tendons.     

Bidirectional regulation of macrophage function by TGF-β

The dual role of transforming growth factor-beta (TGF-β) in modulating macrophage function is an important concept gaining increasing recognition. In addition to its role as a ‘macrophage-deactivating' agent, TGF-β functions as a monocyte activator, inducing cytoke production and mediating host defence. These functions are context-dependent, modulated by the differentiation state of the cell, the local cytokine environment, and the local levels of TGF-β in itself. In general, during the initial stages of inflammation, TGF-β locally acts as a proinflammatory agent by recruiting and activating resting monocytes. As these cells differentiate specific immunosuppressive actions of TGF-β predominate, leading to resolution of the inflammatory response. Increasing our understanding of the bidirectional regulation of macrophage function will facilitate prediction of the ultimate outcome of modulating TGF-β levels in vivo.     

Bone morphogenetic protein-2 and transforming growth factor-β2 interact to modulate human bone marrow stromal cell proliferation and differentiation

Osteoprogenitor cells in the human bone marrow stroma can be induced to differentiate into osteoblasts under stimulation with hormonal and local factors. We previously showed that human bone marrow stromal (HBMS) cells respond to dexamethasone and vitamin D by expressing several osteoblastic markers. In this study, we investigated the effects and interactions of local factors (BMP-2 and TGF-β₂) on HBMS cell proliferation and differentiation in short-term and long-term cultures. We found that rhTGF-β₂ increased DNA content and stimulated type I collagen synthesis, but inhibited ALP activity and mRNA levels, osteocalcin production, and mineralization of the matrix formed by HBMS cells. In contrast, rhBMP-2 increased ALP activity and mRNA levels, osteocalcin levels and calcium deposition in the extracellular matrix without affecting type I collagen synthesis and mRNA levels, showing that rhBMP-2 and rhTGF-β₂ regulate differentially HBMS cells. Co-treatment with rhBMP-2 and rhTGF-β₂ led to intermediate effects on HBMS cell proliferation and differentiation markers. rhTGF-β₂ attenuated the stimulatory effect of rhBMP-2 on osteocalcin levels, and ALP activity and mRNA levels, whereas rhBMP-2 reduced the rhTGF-β₂-enhanced DNA synthesis and type I collagen synthesis. We also investigated the effects of sequential treatments with rhBMP-2 and rhTGF-β₂ on HBMS cell differentiation in long-term culture. A transient (9 days) treatment with rhBMP-2 abolished the rhTGF-β₂ response of HBMS cells on ALP activity. In contrast, a transient (10 days) treatment with rhTGF-β₂ did not influence the subsequent rhBMP-2 action on HBMS cell differentiation. The data show that TGF-β₂ acts by increasing HBMS cell proliferation and type I collagen synthesis whereas BMP-2 acts by promoting HBMS cell differentiation. These observations suggest that TGF-β₂ and BMP-2 may act in a sequential manner at different stages to promote human bone marrow stromal cell differentiation towards the osteoblast phenotype. J. Cell. Biochem. 68:411–426, 1998. © 1998 Wiley-Liss, Inc.     

Transforming growth factor-β enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells

Transforming growth factor-β (TGF-β) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-β₁ on the expression of various plasma membrane determinants. TGF-β₁ induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-β₁-enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-β₁-treated cells with an anti-human β-galactosyltransferase (β-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-β-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, β-GT. These results indicate that TGF-β₁ may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.     

Interleukin-1β and tumor necrosis factor-α have opposite effects on fibroblasts and epithelial cells during basement membrane formation

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.     

Role of the transforming growth factor-β family in the expression of cranial neural crest-specific phenotypes

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-β1 (TGF-β1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-β is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-β antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-β mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-β1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-β is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-β. © 1995 John Wiley & Sons, Inc.     

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

Cultured human melanoma cells were found to secrete TGF-β mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-β in the range from 370 to 610 pg per 10⁶ cells per 24 h. A distinct characteristic of these melanoma cell lines is that they form active surface-bound plasmin by the activation of plasminogen with surface-bound tissue-type plasminogen activator. The present study was performed to assess the role of plasmin in the process of latent TGF-β activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-β present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-β1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-β produced by the melanoma cells was found to inhibit the natural killer cell function of peripheral blood lymphocytes, suggesting that it may have an immunosuppressive effect and a role in the development of melanomas. © 1996 Wiley-Liss, Inc.     

TGF-β1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-β1) inhibits in vitro activation and maturation of DCs. TGF-β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects of TGF-β1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-β1. In contrast to these negative regulatory effects of TGF-β1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-β1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-β1 stimulation for development and function. Recent studies established that TGF-β1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-β1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

Expression,localization, and function of transforming growth factor-βs in embryonic chick spinal cord,hindbrain, and dorsal root ganglia

We have studied the localizations of transforming growth factor-beta (TGF-β) 2 and 3 immunohistochemically using isoform-specific antibodies and TGF-β3 mRNA by in situ hybridization in the nervous system of the 3- to 15-day-old chick embryo with special reference to spinal cord, hindbrain, and dorsal root ganglia (DRG). At embryonic day (E) 3, TGF-β3 mRNA as well as TGF-β2 and 3 immunoreactivities (IRs) were most prominent in the notochord, wall of the aorta, and dermomyotome. At E5 and E7, strong TGF-β2 and 3 IR were seen in or on radial glia of spinal cord and hindbrain. Radial glia in the floor plate region and ventral commissure gave the most intense signal. In the DRG, fiber strands of intense IRs representing extracellular matrix or satellite cells were seen. Neuronal perikarya did not become IR for TGF-β2 and 3 until E11, but even then the moderate signals for TGF-β3 mRNA could not be specifically localized to the neuronal cell bodies. In E11 and older embryos, spinal cord glial or glial progenitor cells, but not neuronal cell bodies were labeled for TGF-β3 mRNA. Immunocytochemistry and western blot analysis indicated that E8 DRG neurons have the TGF-β receptor type II, and treatment of these cells with NGF induces expression of TGF-β3 mRNA. The TGF-β isoforms 1, 2, and 3 did not promote survival of E8 DRG neurons in dissociated cell cultures. All three TGF-β isoforms, however, promoted neurite growth from E8 DRG explants, but were less potent than nerve growth factor. Our data suggest identical localizations of TGF-β2 and -β3 IR in the developing chick and mammalian nervous systems, underscoring the general importance of TGF-βs in fundamental events of neural development. © 1996 John Wiley & Sons, Inc.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Regulation of GM-CSF-Induced Dendritic Cell Development by TGF-β1 and Co-Developing Macrophages

Using a culture system of bone marrow progenitor cells with GM-CSF and TGF-β1, a study was performed to analyze the effect of TGF-β1 on the development of dendritic cells (DC) and to elucidate the regulatory role of macrophages co-developing with dendritic cells. The results demonstrate that DC generated in the presence of TGF-β1 were immature with respect to the expression of CD86, nonspecific esterase activity and cell shape. Such inhibitory effects of TGF-β1 were dependent on FcR⁺ macrophages, which were depleted by panning. TGF-β1 did not appear to inhibit the commitment of progenitor cells to the DC lineage. In addition, TGF-β1 also acted directly on the intermediate stage of DC to prevent their over-maturation, which results in a preferential decrease in MHC class II, but not in CD86, in the presence of TNF-α. FcR⁺ suppressive macrophages were also shown to facilitate DC maturation when stimulated via FcR-mediated signals even in the presence of TGF-β1. These results indicate that TGF-β1 indirectly and directly regulate the development of DC and that co-developing macrophages have a regulatory role in DC maturation.