Identification of a transforming growth factor-β type I receptor transcript in Eriocheir sinensis and its molting-related expression in muscle tissues

The transforming growth factor-β (TGF-β) signaling pathway is conserved across animals, and knowledge of its roles during the molt cycle in crustaceans is presently very limited. This study investigates the roles of the TGF-β receptor in molting-related muscle growth in Eriocheir sinensis. Using the RT-PCR and RACE techniques, we obtained a 1722 bp cDNA sequence encoding a transforming growth factor-β type I receptor in Eriocheir sinensis, designated EsTGFBRI, which contains a 124 bp 5′-untranslated region, a 20 bp partial 3′-untranslated region and a 1578 bp open reading frame encoding 525 amino acids. The deduced EsTGFBRI contains an N-terminal 24 amino acid signal peptide, an activin type I and II receptor domain, a transmembrane helix region, a glycine-serine-rich motif, and a conserved serine/threonine kinase catalytic domain including an activation loop. The qRT-PCR results showed that EsTGFBRI gene was highly expressed in the intermolt testis and ovary in mature crabs. In juvenile crabs, the mRNA levels of EsTGFBRI in claw and abdominal muscles in the later premolt D_3–4 stage were significantly higher than those in the intermolt C and postmolt A–B stages. There was no significant change in EsTGFBRI mRNA levels in walking leg muscles during the molt cycle. The results suggest that EsTGFBRI is probably play roles in molting-related muscle growth in E. sinensis. This study provides a necessary basis for elucidating the functions of TGF-β-like signaling mediated by TGFBRI in molting-related muscle growth in crustaceans.

     

Retinoids potentiate transforming growth factor-β activity in bovine endothelial cells through up-regulating the expression of transforming growth factor-β receptors

Retinoic acid (RA) induces the activation of latent transforming growth factor-β (TGF-β) in bovine aortic endothelial cells (BAECs) via enhancement of cellular plasminogen activator (PA)/plasmin levels. The resultant TGF-β suppresses the excessive fibrinolytic activity by decreasing PA expression and stimulating expression of the PA inhibitor, PA inhibitor-1 (PAI-1), and inhibits cell proliferation. Here, we report that, in this regulatory system, RA simultaneously up-regulates the expression of TGF-β receptor types I and II, resulting in enhancement of TGF-β activity in the cells. RA increased the numbers of high- and low-affinity binding sites for ¹²⁵I-TGF-β1 2.1-fold and 1.5-fold, respectively, without alteration of their Kd values. Affinity labeling and Western and Northern blotting studies showed that, following RA treatment, surface levels of both type I and type II receptors increased due to augmentation in their mRNA levels. The effect was dose- and time-dependent. Treatment with 1 μM RA for 15 hr increased mRNA levels of type I and II receptor threefold and eightfold, respectively. Pretreatment of BAECs with either RA or retinol lowered the concentration of TGF-β1 required to suppress PA levels, to enhance PAI-1 levels, and to inhibit cell proliferation. Thus, retinoids may regulate cellular functions of BAECs not only by inducing the formation of active TGF-β but also by stimulating TGF-β receptor expression. This regulatory mechanism may sustain TGF-β-mediated regulation of EC function at a focal site where RA is acting. J. Cell. Physiol. 176:565–573, 1998. © 1998 Wiley-Liss, Inc.     

The TβR-I pre-helix extension is structurally ordered in the unbound form and its flanking prolines are essential for binding

Transforming growth factor β isoforms (TGF-β) are among the most recently evolved members of a signaling superfamily with more than 30 members. TGF-β play vital roles in regulating cellular growth and differentiation, and they signal through a highly restricted subset of receptors known as TGF-β type I receptor (TβR-I) and TGF-β type II receptor (TβR-II). TGF-β's specificity for TβR-I has been proposed to arise from its pre-helix extension, a five-residue loop that binds in the cleft between TGF-β and TβR-II. The structure and backbone dynamics of the unbound form of the TβR-I extracellular domain were determined using NMR to investigate the extension's role in binding. This showed that the unbound form is highly similar to the bound form in terms of both the β-strand framework that defines the three-finger toxin fold and the extension and its characteristic cis-Ile54-Pro55 peptide bond. The NMR data further showed that the extension and two flanking 3₁₀ helices are rigid on the nanosecond-to-picosecond timescale. The functional significance of several residues within the extension was investigated by binding studies and reporter gene assays in cultured epithelial cells. These demonstrated that the pre-helix extension is essential for binding, with Pro55 and Pro59 each playing a major role. These findings suggest that the pre-helix extension and its flanking prolines evolved to endow the TGF-β signaling complex with its unique specificity, departing from the ancestral promiscuity of the bone morphogenetic protein subfamily, where the binding interface of the type I receptor is highly flexible.     

Transforming growth factor-β receptor expression on human skin fibroblasts: Dimeric complex formation of type I and type II receptors and identification of glycosyl phosphatidylinositol-anchored transforming growth factor-β binding proteins

Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-β (TGF-β) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-β receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-β binding proteins in addition to the type I, II, and III TGF-β receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-β receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-β receptors on these cells. Because the type I receptor does not bind TGF-β in the absence of type II receptor, these results indicate that one molecule of TGF-β induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-β receptors on these cells. These cells respond to TGF-β by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-β receptor signaling paradigm using mutantcells that are unresponsive to TGF-β and cell lines that have been transfected to overexpress these receptors, to include normal TGF-β-responsive cells. In addition, the definition of TGF-β receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases. J. Cell. Physiol. 176:553–564, 1998. © 1998 Wiley-Liss, Inc.     

TGF-β receptor expression on human keratinocytes: A 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with type I and type II receptors

Keratinocytes play a critical role in re-epithelialization during wound healing, and alterations in keratinocyte proliferation and function are associated with the development of various skin diseases. Although it is well documented that TGF-β has profound effects on keratinocyte growth and function, there is a paucity of information on the types, isoform specificity and complex formation of TGF-β receptors on keratinocytes. Here, we report that in addition to the types I, II, and III TGF-β receptors, early passage adult and neonatal human keratinocytes display a cell surface glycosylphosphatidylinositol (GPI)-anchored 150 kDa TGF-β1 binding protein. The identities of the four proteins were confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific anti-receptor antibodies, sensitivity to phosphatidylinositol specific phospholipase C and dithiothreitol, and 2-dimensional electrophoresis. Interestingly, the antitype I TGF-β receptor antibody immunoprecipitated not only the type I receptor, but also the type II receptor and the 150 kDa component, suggesting that the 150 kDa component form heteromeric complexes with the signalling receptors. In addition, two-dimensional (nonreducing/reducing) electrophoresis confirmed the occurrence of a heterotrimeric complex consisting of the 150 kDa TGF-β1 binding protein, the type II receptor, and the type I receptor. This technique also demonstrated the occurrence of types I and II heterodimers and type I homodimers of TGF-β receptors on keratinocytes, supporting the heterotetrameric model of TGF-β signalling proposed using mutant cells and cells transfected to overexpress these receptors. The keratinocytes responded to TGF-β by markedly downregulating all four TGF-β binding proteins and by potently inhibiting DNA synthesis. The demonstration that the 150 kDa GPI-anchored TGF-β1 binding protein forms a heteromeric complex with the TGF-β signalling receptors suggests that this GPI-anchored protein may modify TGF-β signalling in human keratinocytes. J. Cell. Biochem. 70:573–586, 1998. © 1998 Wiley-Liss, Inc.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.     

Pentabromopseudilin: a myosin V inhibitor suppresses TGF-β activity by recruiting the type II TGF-β receptor to lysosomal degradation

Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria Pseudomonas bromoutilis and Alteromonas luteoviolaceus. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-β (TGF-β) activity. PBrP inhibits TGF-β-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-β-induced epithelial–mesenchymal transition in epithelial cells. PBrP inhibits TGF-β signalling by reducing the cell-surface expression of type II TGF-β receptor (TβRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TβRII turnover and the subsequent reduction of TGF-β signalling. Because, TGF-β signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.     

Characterization of a 60-kDa cell surface-associated transforming growth factor-β binding protein that can interfere with transforming growth factor-β receptor binding

We have characterized a 60-kDa transforming growth factor-β (TGF-β) binding protein that was originally identified on LNCaP adenocarcinoma prostate cells by affinity cross-linking of cell surface proteins by using ¹²⁵I-TGF-β1. Binding of ¹²⁵I-TGF-β1 to the 60-kDa protein was competed by an excess of unlabeled TGF-β1 but not by TGF-β2, TGF-β3, activin, or osteogenic protein-1 (OP-1), also termed bone morphogenetic protein-7 (BMP-7). In addition, no binding of ¹²⁵I-TGF-β2 and ¹²⁵I-TGF-β3 to the 60-kDa binding protein on LNCaP cells could be demonstrated by using affinity labeling techniques. The 60-kDa TGF-β binding protein showed no immunoreactivity with antibodies against the known type I and type II receptors for members of the TGF-β superfamily. Treatment of LNCaP cells with 0.25 M NaCl, 1 μg/ml heparin, or 10% glycerol caused a release of the 60-kDa protein from the cell surface. In addition, we found that the previously described TGF-β type IV receptor on GH3 cells, which does not form a heteromeric complex with TGF-β receptors, could be released from the cell surface by these same treatments. This suggests that the 60-kDa protein and the similarly sized TGF-β type IV receptor are related proteins. The eluted 60-kDa LNCaP protein was shown to interfere with the binding of TGF-β to the TGF-β receptors. Thus, the cell surface-associated 60-kDa TGF-β binding protein may play a role in regulating TGF-β binding to TGF-β receptors. J. Cell. Physiol. 173:447–459, 1997. © 1997 Wiley-Liss, Inc.     

Relationship between actions of transforming growth factor (TGF)-β and cell surface expression of its receptors in clonal osteoblastic cells

Various osteoblastic cell lines were examined for the relationship between the presence of cell-surface transforming growth factor (TGF)-β receptors and the synthesis of matrix proteins with their responsiveness to TGF-β. Treatment with TGF-β1 inhibited proliferation and stimulated proteoglycan and fibronectin synthesis in MC3T3-E1 and MG 63 cells. The major proteoglycans synthesized by these cells were decorin and biglycan, and TGF-β1 markedly stimulated the synthesis of decorin in MC3T3-E1 and of biglycan in MG 63 cells. SaOS 2 and UMR 106 cells synthesized barely detectable amounts of decorin or biglycan, and TGF-β1 did not stimulate the synthesis of these proteoglycans. In SaOS 2 cells, however, TGF-β1 enhanced fibronectin synthesis. TGF-β1 did not show any of these effects in UMR 106 cells. Receptor cross-linking studies revealed that only MC3T3-E1 and MG 63 cells had both types I and II signal-transducing receptors for TGF-β in addition to betaglycan. SaOS 2 cells possessed type I but no type II receptor on the cell surface. In contrast, SaOS 2 as well as MC3T3-E1 and MG 63 cells expressed type II receptor mRNA by Northern blot analysis, and cell lysates contained type II receptor by Western blot analysis. Thus, it appears that type II receptor present in SaOS 2 cells is not able to bind TGF-β1 under these conditions. UMR 106 cells with no response to TGF-β1 had neither of the signal-transducing receptors by any of the analyses. These observations using clonal osteoblastic cell lines demonstrate that the ability of osteoblastic cells to synthesize bone matrix proteoglycans is associated with the responsiveness of these cells to TGF-β1, that the responsiveness of osteoblastic cells to TGF-β1 in cell proliferation and proteoglycan synthesis correlates with the presence of both types I and II receptors, and that the effect of TGF-β1 on fibronectin synthesis can develop with little binding of TGF-β1 to type II receptor if type I receptor is present. It is suggested that the combination of cell-surface receptors for TGF-β determines the responsiveness of osteoblastic cells to TGF-β and that changes in cell-surface TGF-β receptors may play a role in the regulation of matrix protein synthesis and bone formation in osteoblasts. © 1995 Wiley-Liss, Inc.     

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.     

Role of receptor complexes in resistance or sensitivity to growth inhibition by TGFβ in intestinal epithelial cell clones

Untransformed rat intestinal epithelial cells (IEC-18) were chemically mutagenized, selected in the presence of TGFβ₁, and cloned by limiting dilution. Two clones (4–5, 4–6) were resistant to growth inhibition by both TGFβ₁ and TGFβ₂. Another clone (4–1) was more sensitive to both TGFβ isoforms (relative to parental IEC-18 cells). IC₅₀ values for TGFβ_{1 and 2} in the 4–1 cells were at least 1/9 those of the parental cells; growth rates were reduced by 49% for TGFβ₁ and by 26% for TGFβ₂ in this clone. This increased sensitivity to TGFβ was explained by the 5- to 10-fold increase, relative to parental cells, in binding of TGFβ₁ and TGFβ₂ to both the type I and II receptors. In contrast, the resistance to growth inhibition by TGFβ in the 4–5 and 4–6 cells could not be explained by a decrease in either TGFβ binding affinities or in total number of receptors expressed, by the presence of serum binding components, or by occupation of receptor binding sites with autocrine TGF-β₁. However, in comparison to TGFβ-sensitive cells (IEC-18, 4–1), the resistant cells displayed a higher ratio of type II relative to type I receptor binding by TGF-β₁. Thus, a critical ratio of binding to receptor subtypes correlated with growth inhibition by TGF-β₁. Resistance to TGF-β₂ in the same clones did not appear to be receptor related. Thus different mechanisms for resistance to TGF-β₁ and TGF-β₂ were observed within a given clone. © 1993 Wiley-Liss, Inc.     

Evidence of Type I and Type II Transforming Growth Factor-β Receptors in Central Nervous Tissues: Changes Induced by Focal Cerebral Ischemia

Abstract: The peptides of the transforming growth factor-β (TGF-β) family transduce their signal through ligand-induced heteromeric complexes that consist of type I and type II serine/threonine kinases. Both TGF-β receptors are abundant in many peripheral tissues, but clear evidence of their expression in cortical astrocytes and neurons has not been published so far. In this study, we investigated the expression of type I and type II TGF-β receptors and their potential ligands (TGF-β1, TGF-β2, and TGF-β3) in the CNS by using RT-PCR and immunohistochemistry. Moreover, to further the study of those cell types that exhibit TGF-β isoforms and related receptors, we examined through the use of RT-PCR whether cortical neurons and astrocytes in culture express the mRNAs for TGF-βs and their receptors. We show that the three TGF-β isoform mRNAs are present in the CNS. However, although astrocytes in culture display all three isoforms, neurons in culture express only TGF-β2. We have demonstrated that both type I and type II TGF-β receptor mRNAs and proteins are present in the CNS and in cultures of cortical neurons and astrocytes. Thus, TGF-βs may act as autocrine and paracrine signals in the CNS between both neurons and astrocytes via the same receptor systems as those found in peripheral tissues. TGF-β1 has been shown to be induced following hypoxic-ischemic brain injury and may play a critical role in the pathophysiology of degenerative processes in the CNS. In the present investigation, we confirmed that the expression of TGF-β1 was increased markedly up until 24 h and thereafter was stable over the first 3 days following permanent occlusion of the middle cerebral artery in mice. However, whereas the expression of the type I TGF-β receptor was not altered by the ischemic insult, the pattern of the type II TGF-β receptors was modified dramatically in the ischemic area 3 days after the occlusion. These data show that, even if ligands are present, they may not be able to transduce their signal. Finally, the present study clearly demonstrates that a knowledge of the expression of ligand-specific receptors following brain injury is a fundamental step in clarifying the involvement of cytokines in neurodegenerative diseases.     

Halofuginone prevents extracellular matrix deposition in diabetic nephropathy

Transforming growth factor-β (TGF-β) is known to promote the accumulation of extracellular matrix (ECM) and the development of diabetic nephropathy. Halofuginone, an analog of febrifugine, has been shown to block TGF-β₁ signaling and subsequent type I collagen production. Here, the inhibitory effect of halofuginone on diabetic nephropathy was examined. Halofuginone suppressed Smad2 phosphorylation induced by TGF-β₁ in cultured mesangial cells. In addition, the expression of TGF-β type 2 receptor decreased by halofuginone. Halofuginone showed an inhibitory effect on type I collagen and fibronectin expression promoted by TGF-β₁. An in vivo experiment using db/db mice confirmed the ability of halofuginone to suppress mesangial expansion and fibronectin overexpression in the kidneys. Moreover, an analysis of urinary 8-OHdG level and dihydroethidium fluorescence revealed that halofuginone reduced oxidative stress in the glomerulus of db/db mice. These data indicate that halofuginone prevents ECM deposition and decreases oxidative stress, thereby suppressing the progression of diabetic nephropathy.     

Tctex3, related to Drosophila Polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6. Received: 10 April 1998 / Accepted: 22 June 1998     

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii^−/−) mice. The effects of arg-ii^−/− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii^−/− cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii^−/− mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.