Activation Of Latent TGF-beta Research Articles

Healing of Burn Wounds in Transgenic Mice Overexpressing Transforming Growth Factor-β1 in the Epidermis

Transforming growth factor-beta (TGF-beta) isoforms are multifunctional cytokines that play an important role in wound healing. Transgenic mice overexpressing TGF-beta in the skin under control of epidermal-specific promoters have provided models to study the effects of increased TGF-beta on epidermal cell growth and cutaneous wound repair. To date, most of these studies used transgenic mice that overexpress active TGF-beta in the skin by modulating the latency-associated-peptide to prevent its association with active TGF-beta. The present study is the first to use transgenic mice that overexpress the natural form of latent TGF-beta 1 in the epidermis, driven by the keratin 14 gene promoter to investigate the effects of locally elevated TGF-beta 1 on the healing of partial-thickness burn wounds made on the back of the mice using a CO(2) laser. Using this model, we demonstrated activation of latent TGF-beta after wounding and determined the phenotypes of burn wound healing. We found that introduction of the latent TGF-beta1 gene into keratinocytes markedly increases the release and activation of TGF-beta after burn injury. Elevated local TGF-beta significantly inhibited wound re-epithelialization in heterozygous (42% closed versus 92% in controls, P < 0.05) and homozygous (25% versus 92%, P < 0.01) animals at day 12 after wounding. Interestingly, expression of type I collagen mRNA and hydroxyproline significantly increased in the wounds of transgenic mice, probably as a result of a paracrine effect of the transgene.

Activation of paracrine TGF-beta1 signaling upon stimulation and degranulation of rat serosal mast cells: a novel function for chymase.

As a source of transforming growth factor beta1 (TGF-beta1), mast cells have been implicated as potential effector cells in many pathological processes. However, the mechanisms by which mast cells express, secrete, and activate TGF-beta1 have remained vague. We show here by means of RT-PCR, immunoblotting, and immunocytochemistry that isolated rat peritoneal mast cells synthesize and store large latent TGF-beta1 in their chymase 1-containing secretory granules. Mast cell stimulation and degranulation results in rapid secretion of the latent TGF-beta1, which is converted by chymase 1 into an active form recognized by the type II TGF-beta serine/threonine kinase receptor (TbetaRII). Thus, mast cells secrete active TGF-beta1 by a unique secretory mechanism in which latent TGF-beta1 and the activating enzyme chymase 1 are coreleased. The activation of latent TGF-beta1 specifically by chymase was verified using recombinant human latent TGF-beta1 and recombinant human chymase. In isolated TbetaRI- and TbetaRII-expressing peritoneal macrophages, the activated TGF-beta1 induces the expression of the plasminogen activator inhibitor 1 (PAI-1), whereas in the mast cells, the levels of TbetaRI, TbetaRII, and PAI-1 expression were below detection. Selective stimulation of mast cells in vivo in the rat peritoneal cavity leads to rapid overexpression of TGF-beta1 in peritoneal mast cells and of TbetaRs in peritoneal macrophages. These data strongly suggest that mast cells can act as potent paracrine effector cells both by secreting active TGF-beta1 and by enhancing its response in target cells.

Participation of thrombospondin-1 in the activation of latent transforming growth factor-beta in malignant glioma cells.

Malignant glioma cells secrete transforming growth factor-beta (TGF-beta) and can activate latent TGF-beta. However, the mechanism of the latent TGF-beta activation has not yet been determined. This study examined whether thrombospondin-1 (TSP-1) secreted by malignant glioma cell lines participates in the activation of latent TGF-beta secreted by the glioma cells. Western blot analysis revealed that TSP-1 was present in both the cell lysates and the culture supernatants of all three malignant glioma cell lines (T98G, A172, and U251). A bioassay for TGF-beta activity revealed that all malignant glioma cell lines used in this study could activate latent TGF-beta by themselves. Latent TGF-beta 1 activation, evaluated by enzyme-linked immunosorbent assay, was inhibited by more than 50% by the addition of neutralizing anti-TSP-1 monoclonal antibody or anti-TSP-1 polyclonal antibody. These results indicate that TSP-1 has a predominant role in the activation of latent TGF-beta in malignant glioma cells.

Latency, activation, and binding proteins of TGF-beta.

The TGF-beta superfamily of growth factors consists of an increasing number of different polypeptide modulators of cell growth, differentiation, and morphogenesis. Three mammalian isoforms have been molecularly cloned. Numerous ways to regulate the expression of the TGF-beta genes have been identified. TGF-betas are, for example, subject to regulation by retinoids, steroid hormones, and vitamin D. A characteristic feature in the biology of TGF-betas is that they are usually secreted from cells in latent forms. The large latent complex consists of the small latent complex (TGF-beta and its propeptide) and a high molecular weight protease resistant binding protein, latent TGF-beta binding protein (LTBP). LTBPs are required for the proper folding and secretion of TGF-beta. TGF-beta is not just secreted from cultured cells but is deposited via LTBPs to the pericellular space, namely to the extracellular matrix. Release of these complexes and activation by proteases is under tight regulation and provides a means to rapidly increase local concentrations of TGF-beta. Biological events, where enhanced or focal proteolysis and activation of latent TGF-beta takes place, include cell invasion, tissue remodeling, and wound healing.

Cooperation of liver cells in health and disease.

Cooperation of liver cells in health and disease.

Activation of latent transforming growth factor-beta1 is induced by mannose 6-phosphate/insulin-like growth factor-II receptor.

This study was conducted to further explore the mechanism of transforming growth factor beta1 (TGF-beta1) activation, which plays a critical role in many physiological and pathological conditions. We have previously shown that the large (270 kDa), but not small (40 kDa), mannose 6-phosphate receptors facilitate the cellular response to latent TGF-beta1 released from genetically modified cells. In this study, we explored the role of cell membrane associated transglutaminase and plasmin in mannose 6-phosphate receptor induced latent TGF-beta activation using MS and MS-9 cells bearing either no receptors or the 270 kDa mannose 6-phosphate/insulin-like growth factor II receptors, respectively. As a source of latent TGF-beta1, PA317 cells were transfected with either pLin-TGF-beta1 vector or pLin retroviral vector with no TGF-beta1 insert using calcium phosphate precipitation. The latency and bioactivity of TGF-beta1 in conditioned medium derived from transfected PA317 cells were evaluated by enzyme-linked immunosorbent assay and mink lung epithelial cell growth inhibition assay, respectively. The level of latent TGF-beta1 was 13-fold higher (20.1 +/- 0.4 vs. 1.5 +/- 0.03 ng/ml) in conditioned medium from pLin-TGF-beta1 transfected cells than that of control. The latency and bioactivity of TGF-beta1 released from pLin-TGF-beta1 transfected cells were confirmed by evaluation of 3H-thymidine incorporation in Mv1Lu epithelial cells treated with non- and heat-activated 10% conditioned medium. The results showed a significantly lower 3H-thymidine incorporation in Mv1Lu epithelial cells treated with heat-activated PA317 conditioned medium (4% of control) relative to those treated with either control or nonheated conditioned medium. This inhibition was abrogated by addition of 40 microg/ml of TGF-beta1 neutralizing antibody. The level of 3H-thymidine incorporation was then evaluated in MS-9 cells receiving Dulbecco's modified Eagle medium containing either 0% 10%, 30% or 50% volumes of nonactivated PA317 conditioned medium for 24 hours. The results showed a markedly lower proliferation in response to 30% and 50% conditioned medium used in MS-9 cells. Under similar experimental conditions, addition of only mannose 6-phosphate, but not fructose 6-phosphate or mannose 1-phosphate, at 1 mM concentration restored the MS-9 cell proliferative response to latent TGF-beta1. The inhibitory effects of latent TGF-beta1 on MS-9 cell proliferation were restored by addition of either TGF-beta1 neutralizing antibody or cystamine, a transglutaminase inhibitor. In contrast, addition of aprotinin, a plasmin inhibitor, had a marginal influence on inhibitory effects of latent TGF-beta1 on MS-9 cell proliferation. Interestingly, a mixture of latent TGF-beta1 + MS-9 cell membranes, but not MS cell membranes, also inhibited the mink lung epithelial cell proliferation (34% of control). These findings indicate that mannose 6-phosphate/insulin-like growth factor II receptors are involved in latent TGF-beta activation and that is at least partly dependent on cell membrane associated transglutaminase, but not on plasmin.

Glucose Stimulation of Transforming Growth Factor-β Bioactivity in Mesangial Cells Is Mediated by Thrombospondin-1

Glucose is a key factor in the development of diabetic complications, including diabetic nephropathy. The development of diabetic glomerulosclerosis is dependent on the fibrogenic growth factor, transforming growth factor-beta (TGF-beta). Previously we showed that thrombospondin-1 (TSP-1) activates latent TGF-beta both in vitro and in vivo. Activation occurs as the result of specific interactions of latent TGF-beta with TSP-1, which potentially alter the conformation of latent TGF-beta. As glucose also up-regulates TSP-1 expression, we hypothesized that the increased TGF-beta bioactivity observed in rat and human mesangial cells cultured with high glucose concentrations is the result of latent TGF-beta activation by autocrine TSP-1. Glucose-induced bioactivity of TGF-beta in mesangial cell cultures was reduced to basal levels by peptides from two different sequences that antagonize activation of latent TGF-beta by TSP, but not by the plasmin inhibitor, aprotinin. Furthermore, glucose-dependent stimulation of matrix protein synthesis was inhibited by these antagonist peptides. These studies demonstrate that glucose stimulation of TGF-beta activity and the resultant matrix protein synthesis are dependent on the action of autocrine TSP-1 to convert latent TGF-beta to its biologically active form. These data suggest that antagonists of TSP-dependent TGF-beta activation may be the basis of novel therapeutic approaches for ameliorating diabetic renal fibrosis.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha- ras -transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Transforming growth factor-β and breast cancer: Mammary gland development

Transforming growth factor (TGF)-β1 is a pluripotent cytokine that profoundly inhibits epithelial proliferation, induces apoptosis, and influences morphogenesis by mediating extracellular matrix deposition and remodeling. The physiologic roles of the action of TGF-β in mammary gland, indeed in most tissues, are poorly understood. In order to understand the actions of TGF-β, we need to take into account the complexity of its effects on different cell types and the influence of context on cellular responses. This task is further compounded by multiple mechanisms for regulating TGF-β transcription, translation, and activity. One of the most significant factors that obscures the action of TGF-β is that it is secreted as a stable latent complex, which consists of the 24-kDa cytokine and the 80-kDa dimer of its prepro region, called latency-associated peptide. Latency imposes a critical restraint on TGF-β activity that is often overlooked.The extracellular process known as activation, in which TGF-β is released from the latent complex, is emphasized in the present discussion of the role of TGF-β in mammary gland development. Definition of the spatial and temporal patterns of latent TGF-β activation in situ is essential for understanding the specific roles that TGF-β plays during mammary gland development, proliferation, and morphogenesis.

The Activation Sequence of Thrombospondin-1 Interacts with the Latency-associated Peptide to Regulate Activation of Latent Transforming Growth Factor-β

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

Keratinocytes suppress transforming growth factor-beta1 expression by fibroblasts in cultured skin substitutes.

Transforming growth factor (TGF)-beta1 is a multipotent growth factor with an important role in tissue homeostasis. This growth factor regulates cell proliferation, adhesion, migration and differentiation, as well as extracellular matrix deposition. The temporal secretion and activation of latent TGF-beta1 is thus of major importance to physiological and pathological processes and in wound healing and tumour formation. Cultured skin substitutes, as used to treat extensive acute or chronic skin wounds, offer an attractive model to investigate cellular interactions in cytokine and growth factor expression and response in vitro. In the present investigation, expression of TGF-beta1 was analysed in keratinocyte, fibroblast and melanocyte monolayer cultures, as well as in the dermal vs. epidermal components of reconstituted human skin. Immunohistology, enzyme-linked immunosorbent assay (ELISA) and Northern blotting were used to demonstrate expression at the RNA and protein level. In the monolayer cultures, levels of TGF-beta1 synthesized by melanocytes were observed to be considerably elevated when compared with keratinocytes. Most TGF-beta1, however, was secreted by fibroblasts. The relative contribution of the epidermal and dermal components of the skin substitutes to overall TGF-beta1 levels was determined by comparing results obtained for either component in the presence and absence of fibroblasts and keratinocytes. From results obtained by ELISA it was apparent that TGF-beta1 levels generated predominantly by fibroblasts within the skin substitutes were greatly reduced over time in the presence of keratinocytes. Suppression of fibroblast TGF-beta1 expression in the presence of keratinocytes was also demonstrable at the RNA level by Northern blotting. Results obtained by immunohistochemistry suggest that most, if not all, of the growth factor was present in the latent form. It is therefore most likely that the observed effect results from a factor secreted by keratinocytes, which is capable of suppressing TGF-beta1 synthesis by fibroblasts. These results suggest that expression of TGF-beta1 by fibroblasts is downregulated by paracrine actions of keratinocytes in healing skin.

Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages.

Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

Characterization of a macrophage-based system for studying the activation of latent TGF-beta.

TGF-beta has been implicated in scarring and tissue fibrosis. Most cells secrete TGF-beta as a high molecular weight, latent complex that must be processed to a lower molecular weight, biologically active form. A number of molecules are involved in this activation step including the mannose 6-phosphate/insulin-like growth factor-II receptor, tissue transglutaminase, thrombospondin, plasmin, and others. Here we describe a rapid macrophage-based system for TGF-beta1 activation, which could be used for screening potential anti-fibrotic agents. The system employs transformed mouse peritoneal macrophages treated with lipopolysaccharide as a cell line capable of activating latent TGF-beta. The activation mechanism in our system involves mannose 6-phosphate/insulin-like growth factor-II receptor and transglutaminase. The activation of latent TGF-beta in this system can be prevented by the addition of mannose-6-phosphate but not mannose-1-phosphate. In addition, transglutaminase inhibitors, antibodies to thrombospondin, insulin-like growth factor-II in the presence of its binding protein IGFBP-2, but not IGFBP-1, suppressed the activation of TGF-beta. Anti-inflammatory molecules, such as hydrocortisone, when added to LPS-treated macrophages, inhibited TGF-beta activation by a mechanism, that may involve downregulation of transglutaminase expression. In summary, this new, rapid and reproducible system allows testing molecules for their ability to inhibit TGF-beta activation, thus providing a screening method for potential anti-scarring molecules.

Active Transforming Growth Factor-β in Wound Repair: Determination Using a New Assay

Transforming growth factor (TGF)-beta regulates wound repair and scarring in an isoform-specific fashion. TGF-beta is produced in a latent form, and its activation is a critical regulatory step controlling the bioactivity of this growth factor. To date, it has been impossible to determine latent TGF-beta activation in vivo due to a lack of quantitative assays. We describe here a semiquantitative modification of the plasminogen activator inhibitor-1/luciferase bioassay (PAI/L assay) for TGF-beta, which we used to determine active and latent TGF-beta isoforms in frozen sections of rat wound tissue. We found that significant amounts of latent TGF-beta were rapidly activated upon wounding (38% of the total TGF-beta at 1 hour after wounding). A second peak of active TGF-beta (17% of total) occurred at 5 days after wounding. The predominant isoforms were TGF-beta1 and -2 with only minor amounts of TGF-beta3 present. This is the first TGF-beta bioassay allowing semiquantitative determination of active and latent isoforms present in vivo, and our results document the significance and temporal regulation of latent TGF-beta isoform activation in wound repair.

Active transforming growth factor-beta in human melanoma cell lines: no evidence for plasmin-related activation of latent TGF-beta.

Cultured human melanoma cells were found to secrete TGF-beta mostly in latent biologically inactive form but in addition five of six melanoma cell lines studied produced in conditioned culture medium active TGF-beta in the range from 370 to 610 pg per 10(6) 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-beta activation in the melanoma cell lines. No direct correlation was found between cell-associated plasmin activity and the amount of active TGF-beta present in the conditioned medium of individual cell lines. The melanoma cell lines exhibited diverse responses to exogenous active TGF-beta 1; three cell lines were growth-stimulated, two were growth-inhibited, and one had a very low sensitivity to the growth factor. The active TGF-beta 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.

Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-beta) as well as a 290 kDa form containing latent TGF-beta binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-beta in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-beta. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-beta1 were found in these digests by TGF-beta1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-beta, in addition to LTBP1 that is associated with the 100 kDa latent TGF-beta complex. These studies show for the first time that latent TGF-beta is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-beta, even at sites distant from the cell, in a relatively avascular environment.

Differential expression and biological activity of retinoic acid-induced TGFbeta isoforms in embryonic palate mesenchymal cells.

The effect of retinoic acid (RA) on TGF-beta mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-beta bioassay in association with TGF-beta isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-beta protein released into the culture medium. RA had little or no effect on TGF-beta1 mRNA expression and protein production. In contrast, RA increased TGF-beta2 and beta3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-beta released was increased relative to the total increase in TGF-beta released, suggesting that RA treatment stimulated activation of latent TGF-beta. RA also increased TGF-beta2 mRNA expression; we have previously shown that RA upregulates TGF-beta3 mRNA in these cells. RA and TGF-beta individually inhibited 3H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit 3H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-beta2 or beta3-specific neutralizing antibodies partially relieved the inhibitory effect on 3H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-beta. Simultaneous treatment with RA and TGF-beta also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-beta treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-beta in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-beta in retinoid-induced cleft palate.

Effect of early luteal phase administration of mifepristone (RU486) on leukaemia inhibitory factor, transforming growth factor beta and vascular endothelial growth factor in the implantation stage endometrium of the rhesus monkey

A single, low-dose administration of a potent antiprogesterone such as mifepristone (RU486) in the early luteal phase results in inhibition of blastocyst implantation in primates. The aim of the present study was to examine the status of leukaemia inhibitory factor (LIF), transforming growth factor beta (TGF beta) and vascular endothelial growth factor (VEGF) in day 6 gestational endometrium of rhesus monkeys with or without exposure to a single dose (2 mg/kg body weight, s.c.) of mifepristone on day 2 after ovulation. Densitometric analyses of immunoblots of endometrial spent media revealed an increase (P < 0.01) in TGF beta pan (TGF beta 1, 2, 3 and 5) and a decrease (P < 0.01) in VEGF secretion from RU486-exposed endometrial samples compared with control samples. Secretory profiles for LIF, TGF beta 1 and TGF beta 1 LAP (latency associated peptide) remained unchanged in the two treatment groups. Morphometric analyses of immunohistochemical staining showed altered cell-specific distribution. TGF beta 1 (P < 0.01) and TGF beta pan (P < 0.02) were higher, while VEGF declined (P < 0.05) in endometrial glands of RU486-exposed endometria compared with control tissue samples. Stromal cell staining patterns for all experimental cytokines studied remained unchanged. In blood vessels, VEGF was found to be low (P < 0.05), while LIF (P < 0.05) and TGF beta 1 (P < 0.01) were higher in mifepristone-exposed endometrial samples compared with control tissue samples. Increased TGF beta secretion together with elevated levels of TGF beta in glandular epithelia and in blood vessels with no apparent change in stromal levels of TGF beta or in levels of TGF beta LAP in any endometrial compartment in the two treatment groups suggest an altered paracrine involvement of this cytokine and an enhanced activation of latent TGF beta in endometrium following mifepristone treatment. Higher levels of TGF beta in gland cells may result in dysregulated growth control and degenerative morphology. Also, higher levels of LIF and TGF beta together with lower levels of VEGF in the vascular compartment in mifepristone-exposed endometrium suggest that endometrial vascular physiology is a target of this anti-progestin during the peri-implantation stage. It is thus plausible that LIF, TGF beta and VEGF in the glandular and vascular compartments of implantation stage endometrium play important roles in rendering the endometrium receptive, and that early luteal phase treatment with an anti-progestin such as mifepristone affects the involvement of these cytokines resulting in endometrial contraception.

Regulation of immune responses by TGF-beta.

The transforming growth factor beta (TGF-beta) family of proteins are a set of pleiotropic secreted signaling molecules with unique and potent immunoregulatory properties. TGF-beta 1 is produced by every leukocyte lineage, including lymphocytes, macrophages, and dendritic cells, and its expression serves in both autocrine and paracrine modes to control the differentiation, proliferation, and state of activation of these immune cells. TGF-beta can modulate expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response, and inhibit them once they have become activated. Increased production and activation of latent TGF-beta have been linked to immune defects associated with malignancy and autoimmune disorders, to susceptibility to opportunistic infection, and to the fibrotic complications associated with chronic inflammatory conditions. In addition to these roles in disease pathogenesis, TGF-beta is now established as a principal mediator of oral tolerance and can be recognized as the sine qua non of a unique subset of effector cells that are induced in this process. The accumulated knowledge gained through extensive in vitro functional analyses and from in vivo animal models, including newly established TGF-beta gene knockout and transgenic mice, supports the concept that clinical therapies based on modulation of this cytokine represent an important new approach to the treatment of disorders of immune function.

Hormonally regulated components of the adrenocortical cell environment and the control of adrenal cortex homeostasis.

The extracellular matrix (ECM) strongly contributes to the regulation of cell proliferation and cell differentiation, and thereby of embryonic development and adult tissue homeostasis. We review here the ongoing characterization of the structure and functions of the extracellular matrix components secreted by adrenocortical cells and discuss their possible implication in the hormonal regulation of adrenal cortex homeostasis. Fibronectin (FN) and laminin (LN) are both major adhesive proteins for adrenocortical cells. FN is synthesized by bovine fasciculata cells in primary culture, and its synthesis is stimulated by TGF(beta)1, TGF(beta)2, and FGF-2 but is not modified by IGF-1 or by the hormones ACTH and angiotensin II. LN is also synthesized by bovine fasciculata cells and its synthesis is specifically stimulated by ACTH. Both proteins are haptotactic and chemotactic for adrenocortical cells, suggesting a physiological role in adrenocyte migration. Their distribution in the adrenal gland is quite distinct. LN is uniformly present in the steroidogenic cells from the three zones, whereas FN is abundant in the fibrovascular structures of the capsule and the cortex. ACTH treatment of adrenocortical cells strongly induces the expression and secretion of thrombospondin-2 (TSP2), a large trimeric matricellular protein. The multimodular structure of TSP2 is the support of a variety of biological functions. TSP2 promotes attachment but prevents spreading of adrenocortical cells. On the other hand, TSP2 induces the activation of latent TGFbeta through an indirect mechanism and is anti-angiogenic in vitro. The overall distribution of TSP2 in the glomerulosa and fasciculata zones of the adrenal cortex, and its absence from the reticularis zone, argue in favor of a role in the protection of adrenocortical cells against apoptosis. In the adrenal cortex, five main biological functions are potentially regulated by components of the extracellular matrix : stem cell commitment into the adrenocyte differentiation pathway, terminal differentiation toward the three distinct adrenocyte phenotypes, centripetal migration, apoptosis and the formation of the capillary network. Future studies will aim at deciphering which extracellular component(s) is involved in each of these regulations.