Expression of scatter factor/hepatocyte growth factor is regionally correlated with the initiation of sperm motility in murine male genital tract: Is scatter factor/hepatocyte growth factor involved in initiation of sperm motility?

Based upon findings that the scatter factor/hepatocyte growth factor (SF/HGF) has strong mitogenic and motogenic properties, and that the sperm cell acquires its fertilizing capacity and motility in the distal parts of mammalian epididymis, the present study was conducted to investigate the role of SF/HGF in initiation of sperm cell motility. This was investigated by determining the expression of SF/HGF in various regions of the murine male genital tract by scatter and cell tracking assays using MDCK epithelial cells, Western blot procedure, and the immunohistochemical procedure using paraffin sections of various regions of the male genital tract. The findings from all these assays indicate that SF/HGF is differentially expressed in various parts of the male genital tract with slight or no expression in the testes, caput epididymis, and vas deferens, and with the highest expression in cauda and corpus (distal) epididymis followed by expression in the corpus (proximal) epididymis. This region-specific SF/HGF expression pattern coincides with the pattern of acquiring the fertilizing capacity and motility by the sperm cell during its transit through the male genital tract. However, wherever SF/HGF was expressed in the male genital tract, its molecular weight was slightly higher (Mr, 82 kD), compared to the SF/HGF expressed in various other somatic tissues (Mr, 78 kD), indicating that the genital tract SF/HGF may be a different molecular species that shares some immunoreactive epitopes with the somatic cell SF/HGF. Incubation of immotile sperm from caput epididymis with the purified human placental SF/HGF of 78 kD initiated motility in 5–15% of sperm population. These results strongly suggest that the SF/HGF-like activity is expressed in the male genital tract in a region-specific manner, and this activity may have a role in initiation of sperm motility acquired during its transit through the epididymis in mammals. © 1994 Wiley-Liss, Inc.     

Platelet-derived growth factor receptor α-positive cells in the tunica muscularis of human colon

An obstacle to understanding motor pathologies of the gastrointestinal (GI) tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is known about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express platelet-derived growth factor receptor α (PDGFRα) in mice and that antibodies for these receptors can be used to label the cells. We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon. Fibroblast-like cells are labelled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. Platelet-derived growth factor receptor α(+) cells are distinct from c-Kit(+) interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or neuronal nitric oxide synthase (nNOS) (inhibitory motor neurons). Platelet-derived growth factor receptor α(+) cells express small conductance Ca(2+)-activated K(+) channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles. Our data suggest that PDGFRα(+) cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help develop an understanding of the role of PDGFRα(+) cells in the human GI tract in health and disease.     

Effects of transforming growth factor β on growth of human mammary epithelial cells in culture

Summary Normal human mammary epithelial cells (HMEC) from different individual reduction mammoplasty specimens were all growth inhibited, and showed a flattened, elongated morphology in response to human recombinant transforming growth factor β1 (TGFβ). The degree of growth inhibition varied among specimens, but none of the normal HMEC maintained growth in the continued presence of TGFβ. The degree of growth inhibition also varied with cell age in vitro, cells closer to senescence being more sensitive. TGFβ sensitivity was additionally assayed in two established cell lines derived from one of the reduction mammoplasty specimens after exposure to benzo(a)pyrene. Although varying degrees of growth inhibition and morphologic changes were observed in the cell lines, both lines contained populations that maintained active growth in the presence of TGFβ. Subclones of these lines demonstrated a great plasticity in their growth response to TGFβ, with individual clones ranging from strongly growth inhibited to nearly unaffected. These results suggest that multiple factors influence the extent of TGFβ-induced growth effects on both normal and transformed mammary epithelial cells, and that some of these factors may act through epigenetic mechanisms. This work was supported by CA24844 from the National Institutes of Health, Bethesda, MD, and the Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-AC03-76SF00098.     

Immunocytochemical localization and concentrations of the α and γ subunits of 7S-nerve growth factor in the submandibular gland of the mouse

Summary For unexplained reasons, nerve growth factor (NGF) exists in very high concentrations in the submandibular gland of the mouse. The NGF in the gland, called 7S-NGF, is a non-covalent complex of three protein subunits, named -, - and -NGF. All the known biological activity resides in the -NGF subunit, and previous studies have shown that -NGF is present in much greater concentrations in the male submandibular gland than in the female gland. The higher concentration in the male is due to the fact that -NGF is synthesized in the granular tubule cells of the submandibular gland. These cells are much more numerous in the male gland.In contrast to -NGF, neither the concentrations of and subunits nor their cellular localization in the mouse submandibular gland have been established. In this study, radioimmunoassays specific for . and subunits determined that both are present in much higher concentrations in the male gland. Immunocytochemical work localized both subunits in the granular tubule cell in the male and female submandibular gland. This indicates that all the components of 7S-NGF exist in a single cell type in the gland and suggests that 7S-NGF can be formed within this cell and secreted as a complex into the saliva.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Immunohistochemical localization of transforming growth factor α in the major salivary glands of male and female rats

Summary The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor (TGF) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF in the rat salivary glands.     

Role of ErbB2 in the prostaglandin E₂-induced enhancement of the mitogenic response to epidermal growth factor in cultured hepatocytes

Prostaglandin E(2) (PGE(2)) enhances the mitogenic response to epidermal growth factor (EGF) in hepatocytes, but the underlying mechanisms are not clear. We previously observed that PGE(2) upregulates EGF-induced signalling in the MEK/ERK and PI3K/Akt pathways in hepatocytes. Other investigations have indicated that ErbB2 enhances the mitogenic effect of EGF in these cells. In the present study we found that treatment with PGE(2) increased ErbB2 and decreased ErbB3 expression at both the mRNA and protein level in cultured rat hepatocytes. Silencing of the ErbB2 expression with specific siRNA blocked the stimulation by PGE(2) and EGF of cyclin D1 expression and DNA synthesis. Both EGF and PGE(2) increased the expression of ERK and Akt, but while the effect of EGF was inhibited by ErbB2-directed siRNA, this did not affect the PGE(2)-induced upregulation of ERK and Akt. These data suggest that PGE(2) can enhance the mitogenic effect of EGF both by increasing ErbB2 expression and by ErbB2-independent mechanisms.     

Transforming growth factor β induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-β induced expression of CTGF was not mediated by TGF-β activated Smad signaling but mediated by TGF-β activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-β activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.     

Transforming growth factor β1 involvement in the conversion of fibroblasts to smooth muscle cells in the rabbit bladder serosa

In an attempt to identify the growth factors or cytokines involved in the serosal thickening that occurs in rabbit bladder subjected to partial outflow obstruction, the following growth factors – transforming growth factor β1, platelet-derived growth factor, epidermal growth factor, granulocyte colony-stimulating factor and granulocyte–monocyte colony-stimulating factor – were delivered separately onto the serosal surface of the intact bladder via osmotic minipumps. The proliferative/differentiative cellular response of the rabbit bladder wall was evaluated by bromodeoxyuridine incorporation and immunofluorescence staining with a panel of monoclonal antibodies to cytoskeletal proteins (desmin, vimentin, keratins 8 and 18 and non-muscle myosin) and to smooth muscle (α-actin, myosin and SM22) proteins. Administration of the transforming growth factor, but not of the other growth factors/cytokines, was effective in inducing serosal thickening. Accumulating cells in this tissue were identified as myofibroblasts, i.e. cells showing a mixed fibroblast–smooth muscle cell differentiation profile. The phenotypic pattern of myofibroblasts changed in a time-dependent manner: 21 days after the growth factor delivery, small bundles of smooth muscle cells were found admixed with myofibroblasts, as occurs in the obstructed bladder. These ‘ectopic’ muscle structures displayed a variable proliferating activity and expressed an immature smooth muscle cell phenotype. The complete cellular conversion to smooth muscle cells was not achieved if transforming growth factor β1 was delivered to fibroblasts of subcutaneous tissue. These findings suggest a tissue-specific role for this growth factor in the cellular conversion from myofibroblast to smooth muscle cells. © 1998 Chapman & Hall     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

Developmental expression of transforming growth factor-α and epidermal growth factor receptor proteins in the human pancreas and digestive tract

This study was designed to localize transforming growth factor alpha (TGF-) and epidermal growth factor receptor (EGFR) expression in the developing human gastrointestinal tract and pancreas. Immunohistochemical techniques using specific antibodies against human TGF- and EGFR were performed on digestive tissues of fetuses from 9 to 10 to 24 weeks of gestation, children and adults. In fetuses, TGF- and EGFR proteins were expressed in all epithelial tissues studied with a good correlation and from an age as early as 9 to 10 weeks of gestation, except for TGF- in the esophagus. The strongest TGF- immunostaining was noted in the stomach and the proximal colon. Unexpectedly, immunoreactive gut endocrine cells were observed with the two antibodies used. Relatively numerous in fetuses, they decreased in number with age and were rare in adults particularly along the colon. Enteroglucagon-secreting cells were shown to express TGF- while some gastrin, somatostatin and pancreatic glucagon cells were immunostained with EGFR antibodies. The presence of TGF- and of its recetor in digestive tract epithelium and pancreatic tissues early in fetal life suggests a functional role for TGF- during the developmental process of the digestive system. We demonstrate that TGF- is also produced by endocrine cells and might have an additional mode of action other than paracrine, at least during fetal life.     

Roles of insulin-like growth factor Ⅱ in regulating female reproductive physiology

The number of growth factors involved in female fertility has been extensively studied, but reluctance to add essential growth factors in culture media has limited progress in optimizing embryonic growth and implantation outcomes, a situation that has ultimately led to reduced pregnancy outcomes. Insulin-like growth factor Ⅱ(IGF-Ⅱ) is the most intricately regulated of all known reproduction-related growth factors characterized to date, and is perhaps the predominant growth factor in human ovarian follicles. This review aims to concisely summarize what is known about the role of IGF-Ⅱ in follicular development, oocyte maturation, embryonic development, implantation success, placentation, fetal growth, and in reducing placental cell apoptosis, as well as present strategies that use growth factors in culture systems to improve the developmental potential of oocytes and embryos in different species. Synthesizing the present knowledge about the physiological roles of IGF-Ⅱ in follicular development, oocyte maturation, and early embryonic development should, on the one hand, deepen our overall understanding of the potential beneficial effects of growth factors in female reproduction and on the other hand support development(optimization) of improved outcomes for assisted reproductive technologies.     

The role of transforming growth factor (TGF)-β in modulating the immune response and fibrogenesis in the gut

Transforming growth factor (TGF)-β, a pleiotropic cytokine released by both immune and non-immune cells in the gut, exerts an important tolerogenic action by promoting regulatory T cell differentiation. TGF-β also enhances enterocyte migration and regulates extracellular matrix turnover, thereby playing a crucial role in tissue remodeling in the gut. In this review we describe the mechanisms by which abnormal TGF-β signaling impairs intestinal immune tolerance and tissue repair, thus predisposing to the onset of immune-mediated bowel disorders, such as inflammatory bowel disease and celiac disease. Additionally, we will discuss potential therapeutic strategies aiming at restoring physiologic TGF-β signaling in chronic intestinal diseases.     

Expression of human transforming growth factor β1 in E. coli (I) --Direct expression in cytoplasm

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Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.