Motogenic and biosynthetic response of adult skin fibroblasts to TGF-beta isoforms (-1, -2 and -3) determined by 'tissue response unit': role of cell density and substratum

We have recently demonstrated that the three principal mammalian isoforms of transforming growth factor beta (TGF-beta) exert distinct effects upon: (1) the migration of confluent adult fibroblasts into 3D gels of native type I collagen fibres (i.e. TGF-beta-1 and -2 had no apparent motogenic activity, whilst TGF-beta-3 induced a dose-dependent stimulation of cell migration); and (2) the synthesis of hyaluronan (HA) by these cells is also affected by the TGF-beta isoforms in a manner which parallels their effect on cell migration. The objective of the present study is to elucidate the manner in which this differential activity of the TGF-beta-1, -2 and -3 may be modulated by experimental parameters. Data presented in this communication indicate that cytokine bioactivity is determined by a combination of cell density and the nature of the macromolecular substratum. Thus, we now report that all three TGF-beta isoforms inhibit the migration of subconfluent cells in the collagen gel assay. Our data confirm that the migration of confluent cells is stimulated by TGF-beta-3 and further indicate that this motogenic activity is completely abrogated by either TGF-beta-1 or -2 when these are co-incubated with TGF-beta-3. In contrast to these results obtained using a native type I collagen substratum, all three isoforms stimulated adult fibroblast migration in the transmembrane assay (in which cells are adherent to a 2-D porous polycarbonate substratum). The precise effect of TGF-beta isoforms on HA synthesis was also affected by cell density and the nature of the substratum in a manner which paralleled their diverse effects on cell migration (i.e. stimulation, inhibition or no effect). Streptomyces hyaluronidase completely neutralized the TGF-beta-3-induced stimulation of confluent fibroblast migration, thus suggesting a mechanistic link between the cytokine-induced cell migration and HA synthesis under these conditions. Taken together, these data indicate that: (1) the bioactivity of TGF-beta-1, -2 and -3 are determined by cell density, the macromolecular substratum and the presence of other cytokines; and (2) it is therefore necessary to define cytokine bioactivity within the context of a larger 'tissue response unit' which more fully defines the activity state of the target cell and its microenvironment.     

Regulatory effects of platelet-derived growth factor-AA homodimer on migration of vascular smooth muscle cells.

Migration of medial smooth muscle cells (SMC) into the intima is important in intimal thickening of atherosclerotic tissues. To study the functions of three isoforms of platelet-derived growth factor (PDGF) in atherosclerosis, we investigated their effects on SMC migration by Boyden's chamber method. Although PDGF-AB and PDGF-BB enhanced SMC migration dose-dependently, PDGF-AA did not enhance SMC migration, but instead inhibited SMC migration induced by PDGF-AB or PDGF-BB. PDGF-AA also inhibited SMC migration induced by two other migration factors, fibronectin and SMC-derived migration factor. PDGF-AA is considered to be coexpressed with transforming growth factor (TGF)-beta 1 in atherosclerotic tissues. Treatment of SMC with TGF-beta 1 reduced an autocrine migration activity from SMC. Studies using anti-PDGF antibody revealed that an increased secretion of PDGF-AA by TGF-beta 1 caused the reduced migration activity. cAMP increase by forskolin and dibutyryl cAMP suppressed SMC migration, whereas cAMP decrease by pertussis toxin had no effects on PDGF-AA-suppressed migration. In contrast, staurosporine, an inhibitor of protein kinase C, enhanced SMC migration and neutralized the inhibitory effect of PDGF-AA. These findings suggest that PDGF-AA regulates SMC migration in intimal thickening in atheroma formation and that protein kinase C may play an important role in the inhibitory mechanism of PDGF-AA.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Release of transforming growth factor-beta 1 from the pericellular matrix of cultured fibroblasts and fibrosarcoma cells by plasmin and thrombin.

A sensitive immunoblotting assay was developed for the detection of transforming growth factor (TGF)-beta 1 from cell extracts and culture medium. HT-1080 human fibrosarcoma cells and human fibroblasts were used as models for the secretion and proteolytic release of pericellular matrix-associated TGF-beta 1. Analysis of the pericellular matrices of the cells indicated that the majority of cell-layer associated TGF-beta 1 was associated with the pericellular matrix. Treatment of the cells with plasmin or thrombin released the matrix-associated TGF-beta 1 to the culture medium. Assays for the biological activity of plasmin-released TGF-beta 1 by Mv1Lu cell growth inhibition assays indicated that the majority was in the latent form. Northern hybridization analyses indicated that the mRNA levels of TGF-beta 1 were not elevated during the proteinase treatment. Experiments using radiolabeled TGF-beta 1 indicated that exogenous active TGF-beta 1 associates mainly with the presumed TGF-beta 1 receptors that were not retained in the extracellular matrix preparations. These results indicate that a major fraction of latent TGF-beta 1 that is produced by the cells is deposited to and remains associated with the pericellular matrices of cultured fibroblasts and fibrosarcoma cells, and that matrix-associated TGF-beta 1 is very susceptible to release by various proteolytic enzymes.     

Transforming growth factor-beta2 enhances differentiation of cardiac myocytes from embryonic stem cells

Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-beta (TGF-beta) -beta1, -beta2, and -beta3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-beta1, or -beta3, treatment of mouse ES cells with TGF-beta2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-beta2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and alpha-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-beta2 but not TGF-beta1, or -beta3 promotes cardiac myocyte differentiation from ES cells.     

Regulation of proliferation and migration in retinoic acid treated C3H10T1/2 cells by TGF-beta isoforms

Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells. In conclusion: (1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.     

Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.

Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.     

Fibrogenesis. V. TGF-beta signaling pathways

Transforming growth factor (TGF)-beta is a multifunctional peptide growth factor with a wide range of potential effects on growth, differentiation, extracellular matrix deposition, and the immune response. General TGF-beta signaling pathways have been described in detail over the last several years, but factors that determine the nature of the TGF-beta response are poorly understood. In particular, signaling pathways that specifically mediate the matrix effects of TGF-beta have received little attention, although they will be important therapeutic targets in the treatment of pathological fibrosis. This themes article focuses on TGF-beta signaling and highlights potential points for generating matrix-specific responses.     

Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands.Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.     

The secretory beta-amyloid precursor protein is a motogen for human epidermal keratinocytes

Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the beta-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed beta1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.     

Neutral endopeptidase-24.11 (NEP) deactivates PDGF- and TGF-beta-induced cell shape changes in invertebrate immunocytes

Using a cytofluorimetric assay, we found that immunocytes of the mollusc, Mytilus galloprovincialis, express CD10, a surface antigen known to be identical to neutral endopeptidase-24.11 (NEP). The spectrofluorimetric analysis demonstrates that the growth factors PDGF-AB and TGF-beta1 provoke an increase in NEP-like activity in membrane preparations from the immunocytes, but have no effect on the soluble form in the serum. On the other hand, computer-assisted microscopic image analysis reveals that NEP deactivates the PDGF-AB- and TGF-beta1-induced shape changes in immunocytes. However, Western blots show that, in solution, NEP does not cleave PDGF-AB or TGF-beta1, indicating that the inactivation is not due to proteolysis. These results suggest a functional interplay in invertebrate immunocytes between growth factors and NEP, as previously shown in vertebrate cells.     

Expression of TGF-beta signaling genes in the normal, premalignant, and malignant human trophoblast: loss of smad3 in choriocarcinoma cells

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.     

Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which factors control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming growth factor)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral fibroblasts. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral fibroblasts, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately 15% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye growth.     

Downregulated expression of integrin alpha6 by transforming growth factor-beta(1) on lens epithelial cells in vitro

Integrins represent the main cell surface receptors that mediate cell-matrix and cell-cell interactions. They play critical roles in adhesion, migration, morphogenesis, and the differentiation of several cell types. Previous studies have demonstrated that members of the fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-beta(1), and insulin growth factor (IGF)-1 play important roles in lens biology. In particularly, TGF-beta(1) appears to play a key role in extracellular matrix production, cell proliferation, and cell differentiation of lens epithelial cells. In this study we investigated the effects of FGF-2, TGF-beta(1), and IGF-1 on the modulation of integrin receptors using lens epithelial cell lines (HLE B-3 and alphaTN-4) and lens explants. We found that the expression of integrin alpha6 is downregulated by TGF-beta(1), but is not responsive to FGF-2 or IGF-1. The promoter activity of the integrin alpha6 gene decreased upon TGF-beta(1) treatment in a transient transfection assay, and flow cytometric analysis demonstrated the reduced expression of integrin alpha6 by TGF-beta(1), whereas significant changes were not observed in the level of integrin alpha6 after the addition of FGF-2. These findings suggest that the reduced expression of integrin alpha6 caused by TGF-beta(1) might play a role in the activation of the cell cycle genes required during the fiber differentiation of the lens.     

Discordant transforming growth factor beta 1 RNA and protein localization during chemical carcinogenesis of the skin.

Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.     

Stress-relaxation and contraction of a collagen matrix induces expression of TGF-beta and triggers apoptosis in dermal fibroblasts.

Extracellular matrix serves as a scaffold for cells and can also regulate gene expression and ultimately cell behaviour. In this study, we compared the effects of three forms of type I collagen matrix, which differed only in their mechanical properties, and plastic on the expression of transforming growth factor-beta1 (TGF-beta1), matrix metalloproteinase-1 (collagenase), and type I collagen and on the growth and survival of human dermal fibroblasts. These effects were correlated with alterations in cell morphology and organization of intracellular actin. Cells in detached or stress-relaxed matrices were spherical, lacked stress fibres, and showed increased TGF-beta1 mRNA compared to the cells in anchored collagen matrices or on plastic, which were polygonal or bipolar and formed stress fibres. The levels of TGF-beta measured by bioassay were higher in detached and stress-relaxed collagen matrices, than in anchored collagen matrices. Cells on plastic contained little or no immunoreactive TGF-beta, while most cells in collagen matrices were stained. The levels of collagenase mRNA were significantly higher in all the collagen matrix cultures compared to those on plastic, but there were no statistically significant differences between them. Levels of mRNA for procollagen type I were not significantly affected by culture in the collagen matrices. Apoptotic fibroblasts were detected by the TUNEL assay in detached (5.7%) and to a lesser extent in stress-relaxed (2.2%) matrices, but none were observed in anchored collagen matrices or on plastic. These results show that alterations in the mechanical properties of matrix can induce the expression of TGF-beta and trigger apoptosis in dermal fibroblasts. They further suggest that inability to reorganize this matrix could be responsible for the maintenance of the fibroproliferative phenotype associated with fibroblasts in hypertrophic scarring.     

Progressive pulmonary fibrosis is mediated by TGF-beta isoform 1 but not TGF-beta3

Tissue repair is a well-orchestrated biological process involving numerous soluble mediators, and an imbalance between these factors may result in impaired repair and fibrosis. Transforming growth factor (TGF)-beta is a key profibrotic element in this process and it is thought that its three isoforms act in a similar way. Here, we report that TGF-beta3 administered to rat lungs using transient overexpression initiates profibrotic effects similar to those elicited by TGF-beta1, but causes less severe and progressive changes. The data suggest that TGF-beta3 does not lead to inhibition of matrix degradation in the same way as TGF-beta1, resulting in non-fibrotic tissue repair. Further, TGF-beta3 is able to downregulate TGF-beta1-induced gene expression, suggesting a regulatory role of TGF-beta3. TGF-beta3 overexpression results in an upregulation of Smad proteins similar to TGF-beta1, but is less efficient in inducing the ALK 5 and TGF-beta type II receptor (TbetaRII). We provide evidence that this difference may contribute to the progressive nature of TGF-beta1-induced fibrotic response, in contrast to the limited fibrosis observed following TGF-beta3 overexpression. TGF-beta3 is important in "normal wound healing", but is outbalanced by TGF-beta1 in "fibrotic wound healing" in the lung.     

Autocrine regulation of T cell motility by calreticulin-thrombospondin-1 interaction

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.     

An improved recombinant mammalian cell expression system for human transforming growth factor-beta2 and -beta3 preparations

Transforming growth factor-beta2 and -beta3 (TGF-beta2 and -beta3) are important members of TGF-beta family which play important roles in the growth, maintenance, and repair processes of developing embryos, neonates, and adults. Preparation of large quantities of these two cytokines, which is necessary for structural studies and other applications, has proven to be extremely difficult. We have developed a novel Chinese hamster ovary cell-based expression system for high-level expression and high recovery of recombinant human TGF-beta2 and -beta3. In this system, we used a mammalian expression vector which contains a glutamine synthetase coding region for amplification, together with a modified TGF-beta2 or -beta3 open reading frame for expression. The leader peptide of TGF-beta2 or -beta3 was replaced by that from the V-J2-C region of a mouse immunoglobulin kappa-chain, and a poly-histidine tag was inserted immediately after the leader sequence to facilitate protein purification without changing the mature TGF-beta2 or -beta3 amino acid sequence. In addition, the extreme N-terminal cysteine residue of TGF-beta2 or -beta3 was replaced by a serine residue. The resulting expression constructs produced two stable cell clones expressing 10 mg of TGF-beta2 and 8 mg of TGF-beta3 per liter of spent medium. The purification scheme involved the use of two simple chromatographic steps with a typical yield of 5 mg of TGF-beta2 and 4 mg of TGF-beta3. This method represents a significant improvement over previously published methods and may be applicable to other TGF-beta superfamily members. We further confirmed that latent TGF-beta2 and -beta3 can be activated by proteolysis and glycolysis, which have not been reported before.     

TGF-beta and the regulation of neuron survival and death.

Transforming growth factor-betas (TGF-betas) constitute a superfamily of multifunctional cytokines with important implications in morphogenesis, cell differentiation, and tissue remodeling. In the developing nervous system, TGF-beta2 and -beta3 occur in radial and astroglial cells as well as in many populations of postmitotic, differentiating neurons. TGF-beta1 is restricted to the choroid plexus and meninges. In addition to functions related to glial cell maturation and performances, TGF-beta2 and -beta3 are important regulators of neuron survival. In contrast to neurotrophic factors, as for example, neurotrophins, TGF-betas are most likely not neurotrophic by themselves. However, they can dramatically increase the potency of select neurotrophins, fibroblast growth factor-2, ciliary neurotrophic factor, and glial cell line-derived neurotrophic factor (GDNF). In the case of GDNF, we have shown that GDNF fails to promote the survival of highly purified neuron populations in vitro unless it is supplemented with TGF-beta. This also applies to the in vivo situation, where antibodies to all three TGF-beta isoforms fully prevent the trophic effect of GDNF on axotomized, target-deprived neurons. In addition to the TGF-beta isoforms -beta2 and -beta3, other members of the TGF-beta superfamily are expressed in the nervous system having important roles in embryonic patterning, cell migration, and neuronal transmitter determination. We have cloned and expressed a novel TGF-beta, named growth/differentiation factor-15 (GDF-15). GDF-15 is synthesized in the choroid plexus and released into the CSF, but also occurs in all regions investigated of the developing and adult brain. GDF-15 is a potent trophic factor for developing and 6-OHDA-lesioned midbrain dopaminergic neurons in vitro and in vivo, matching the potency of GDNF.