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

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

Differential display of human marrow stromal cells reveals unique mRNA expression patterns in response to dexamethasone

Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic‐supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10⁻⁸ M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up‐regulated and down‐regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor‐β‐induced gene product (βig‐h3), calphobindin II, cytosolic thyroid‐binding protein, 22‐kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady‐state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the βig‐h3 gene, which showed a relative 10‐fold decrease in mRNA levels after 6 days of treatment. Interestingly, βig‐h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX‐treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231–243, 1999. Published 1999 Wiley‐Liss, Inc.     

TGF-β: from latent to active

The transforming growth factor-betas (TGF-βs) are synthesized as precursor proteins that are modified intracellularly prior to secretion. One of the most relevant intracellular modifications is the cleavage of the C-terminal pro-region from the N-terminal portion of the protein. The C-terminal pro-region is referred to as the latency-associated peptide (LAP) while the N-terminal region is called the mature TGF-β or active TGF-β. However, with some exceptions the LAP noncovalently associates with the mature TGF-β prior to secretion. When the mature TGF-β is associated with the LAP it is called L-TGF-β and cannot interact with its receptor and has no biological effect. The TGF-βs and their receptors are very ubiquitously expressed, suggesting that the regulation of TGF-β activity is likely to be complex and multifactorial. However, one of the most important means of controlling the biological effects of TGF-β is the regulation of converting L-TGF-β to active TGF-β. The current literature supports two major mechanisms of activation of L-TGF-β and suggests that the mechanism of activation of L-TGF-β may be varied and context-dependent. For TGF-β to become biologically active the LAP has to be either released from its associations with L-TGF-β or undergo conformational change such that the LAP is not released from the L-TGF-β complex but exposes the TGF-β receptor binding site. Since TGF-β has been associated with the pathogenesis of numerous diseases, the various mechanisms of activation of L-TGF-β in context offer the possibility of controlling TGF-β activity localized to the organ of involvement and to a more specific disease process.     

Effects of transforming growth factors and activin-A on in vitro porcine oocyte maturation

Growth factors are known to regulate ovarian function. In the present study, effects of these growth factors, TGF-α, TGF-β, and activin-A were tested on spontaneous porcine oocyte maturation. Cumulus-oocyte complexes (COC) were cultured in the presence of TGF-α, TGF-β, and activin-A for 48 hr. Stages of meiotic maturation were assessed by staining with acetic orcein. Among these factors, only TGF-α significantly enhanced the maturation rate, whereas TGF-β suppressed the spontaneous maturation rate. The site of action of TGF-α on COC and the interaction between TGF-α and EGF receptor was also examined. Denuded oocytes, alone or in coculture with cumulus cells, were cultured in the presence of TGF-α for 48 hr. TGF-α did not have any significant effect on denuded oocyte maturation. Heptanol was employed to investigate the role of gap junctions on TGF-α-induced oocyte maturation in COC. Although heptanol did not have any significant effect in the control medium, heptanol reversed the stimulatory effect of TGF-α on porcine oocyte maturation. TGF-α was able to displace ¹²⁵I-EGF binding on COC. In conclusion, TGF-α enhances the spontaneous maturation of porcine oocytes by generating positive signal(s) in cumulus cells that are transferred to the oocyte via gap junctions. TGF-α shares the same receptor with EGF on porcine COC. TGF-β, in contrast, inhibits porcine oocyte maturation. © 1994 Wiley-Liss, Inc.     

TGF-β and cancer

The relationships between transforming growth factor-β (TGF-β) and cancer are varied and complex. The paradigm that is emerging from the experimental evidence accumulated over the past decade or so is that TGF-β can play two different and opposite roles with respect to the process of malignant progression. During early stages of carcinogenesis, TGF-β acts predominantly as a potent tumor suppressor and may mediate the actions of chemopreventive agents such as retinoids and nonsteroidal anti-estrogens. However, at some point during the development and progression of malignant neoplasms, bioactive TGF-βs make their appearance in the tumor microenvironment and the tumor cells escape from TGF-β-dependent growth arrest. In many cases, this resistance to TGF-β is the consequence of loss or mutational inactivation of the genes that encode signaling intermediates. These include the types I and II TGF-β receptors, as well as receptor-associated and common-mediator Smads. The stage of tumor development or progression at which TGF-β-resistant clones come to dominate the tumor cell population in different types of neoplasm remains to be defined. The phenotypic switch from TGF-β-sensitivity to TGF-β-resistance that occurs during carcinogenesis has several important implications for cancer prevention and treatment.     

TGF-β in infections and infectious diseases

Since it was first described as having the ability to inhibit macrophage activation, transforming growth factor-beta (TGF-β) has been analyzed for its role in regulating immune responses to a variety of pathogens, including viruses, bacteria, yeast, and protozoa. Most of the studies have involved organisms that infect macrophages, and this discussion will attempt to highlight these findings. Perhaps the most work has been performed with protozoan pathogens, including Trypanosoma cruzi and a variety of Leishmania species, so the discussion will begin with these organisms. Other studies have focused on mycobacteria and viruses, including human immunodeficiency virus, so these areas will also be emphasized in the discussion. For the most part, investigators have reported that TGF-β has, as expected, a negative influence on host responses and a beneficial effect on the survival and growth of intracellular pathogens. However, other studies have found that TGF-β may have a positive or beneficial effect in some models of infection. This review will attempt to highlight studies and conclusions on the roles of TGF-β in infection.     

TGF-β: 20 years and counting

The history of transforming growth factor-beta (TGF-β) as a bifunctional agent in the immune system is briefly described. The importance of cellular context in understanding the role of TGF-β in regulating immune response is emphasized.     

Role of NK cells and TGF-β in the regulation of T-cell-dependent antibody production in health and autoimmune disease

Natural killer (NK) cells are a third lymphocyte population especially important in innate immunity. NK cells may also have an important role in the regulation of acquired immunity. These lymphocytes spontaneously produce large amounts of both active and latent transforming growth factor-beta (TGF-β). NK-cell-derived TGF-β1 enabled activated CD8⁺ T cells to inhibit antibody production by blocking the induction of this response. Production of lymphocyte-derived TGF-β is decreased in systemic lupus erythematosus. Insufficient levels of this cytokine in SLE and other autoimmune diseases may contribute to defective T regulatory cell function characteristic of this and other autoimmune diseases. NK cells are found in mucosal tissues and the TGF-β spontaneously released by these cells could contribute to the usual tolerogenic response of T cells to antigens presented at these sites. Thus, in addition to its well known immunosuppressive effects, TGF-β could have an equally important role in the generation of regulatory T cells.     

TGF-β1 is an organizer of responses to neurodgeneration

TGF-β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF-β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF-β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF-β1 protein observed in βA/amyloid-containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF-β1 in these human brain disorders is discussed in relation to the potent effects of TGF-β1 on wound healing and inflammatory responses in peripheral tissues. We hypothesize that TGF-β1 and possibly other TGF-β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non-neural tissues. Work from many laboratories has shown that activities of TGF-β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF-β's are well established in other tissues.     

TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-beta1 (TGF-beta1)-induced pulmonary fibroblast-myofibroblast differentiation to examine the role of the TGF-beta1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-beta1 induced marked alteration in cell phenotype, such that cells resembled "epithelioid-postmitotic fibroblasts." This was associated with marked reorganization of the actin cytoskeleton and upregulation of alphaSMA gene expression. TGF-beta1 stimulation also induced alphaSMA protein expression with increased incorporation of alphaSMA into stress fibers. Following stimulation with TGF-beta1, subsequent addition of serum-free medium did not reverse TGF-beta1-induced morphological change, suggesting that TGF-beta1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFbeta1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-beta1 independent alteration in fibroblast cell phenotype, up-regulation of alphaSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-beta1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.     

Sequential deposition of latent TGF-β binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts

Latent TGF-beta binding proteins (LTBPs) mediate the targeting of latent TGF-beta complexes into ECM structures, which is important for TGF-beta activation and functions. LTBPs-1, -3 and -4 associate with and regulate the bioavailability of TGF-betas. We investigated whether LTBP-3 and -4 are associated with pericellular fibrillar structures of human lung fibroblast ECM, and which of their domains are important for this function. Immunoblotting analyses of isolated insoluble matrices as well as immunofluorescence analyses and confocal microscopy indicated that both LTBP-3 and -4 get assembled into the ECM. Interestingly, LTBP-4 was not detected until 7-10 days of culture and LTBP-3 until 14 days of culture. This was a major difference from the deposition kinetics of LTBP-1, which was detected already within 2 days of culture. Expression analyses by real time RT-PCR indicated that the slow appearance of LTBP-3 and -4 was due to the low expression levels soon after subculture. Recombinant N-terminal fragments of LTBP-3 and -4 bound readily to fibroblast ECM. The C-terminal domain of LTBP-4, but not of LTBP-3, also associated with the matrix structures. The levels of ECM-associated latent complexes of TGF-beta1 increased in parallel with the increased production and deposition of the LTBPs. The amount of active TGF-beta in the conditioned medium decreased during extended culture. Our results suggest that ECM is an important site of deposition also for LTBP-3 and -4 and that the temporal and spatial targeting of the TGF-beta complexes are associated with ECM maturation.     

TGF-β1: immunosuppressant and viability factor for T lymphocytes

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine with multiple roles in the immune system. To date, it has been difficult to develop a comprehensive picture of the effect of TGF-β on T lymphocytes, because TGF-β not only acts directly on T lymphocytes, but also acts indirectly by regulating the function of antigen-presenting cells. In early studies, it was mostly the inhibitory function of TGF-β that was demonstrated; recently, however TGF-β was recognized as an antiapoptotic survival factor for T lymphocytes. The outcome of the TGF-β effect on T lymphocytes was shown to strongly depend on their stage of differentiation and on the cytokine milieu. TGF-β cannot be classified as a classical Th1 or Th2 cytokine. However, recently the existence of the TGF-β-producing Th3 subset was described which might play an important regulatory role during an immune response. A better understanding of the molecular mechanism of how TGF-β inhibits or stimulates T lymphocytes will help to predict the complex functions of this cytokine.     

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis

Gene deletion experiments have shown that the three TGFβ isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins αvβ6 and αvβ8 activate latent forms of TGFβ1 and TGFβ3. This raises the possibility that TGFβ1 and TGFβ3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFβ1 activation (Tgfb1^RGE/RGE) that were also homozygous for a null mutation in the TGFβ3 gene. Tgfb1^RGE/RGE; Tgfb3^−/− mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking αvβ8. Some Tgfb1^RGE/RGE; Tgfb3^+/− and Tgfb1^RGE/RGE; Tgfb3^+/+ mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFβ1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3^−/− mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFβ1 and TGFβ3 during development based on a shared mechanism of activation.