Transforming Growth Factor-ß|
Multiple Actions and Potential Clinical Applications
Michael B. Sporn, MD, Anita B. Roberts, PhD
THE CURRENT excitement in research on transforming growth factor-ß (TGF-ß) comes from its multiple actions on almost every type of cell and its potential for therapeutic use in common clinical conditions for which there are no adequate pharmacologic agents. Although TGF-ß was identified originally in an assay that measured its ability to enhance the growth of fibroblasts in soft agar ("transformation"), its true importance is as a mediator of nomal cellular physiology, in particular during normal formation of tissues (as in embryogenesis) and during tissue response to injury (as in inflammation and repair). Almost all cells have been shown to make TGF-ß in one of its molecular forms, and almost all normal cells have receptors for TGF-ß.
Transforming growth factor-ß 1 is a highly stable peptide that consists of two identical chains, each containing 112 amino acids. It was first isolated and characterized from human platelets and placentas as well as from bovine kidneys, and then was cloned from a human complementary DNA- library. The universality of its action is emphasized by the fact that its amino acid sequence is identical in man, monkeys, cows, pigs and chickens. Four additional, closely related TGF-ßs have been identified subsequently and their separate genes cloned. The cloning of these four new genes will allow large-scale production of their respective peptides by recombinant DNA-technology, as already has occured with TGF-ß 1.
Transforming growth factor-ß is highly pleiotropic. It can stimulate proliferation in some cells, especially in connective tissue, while being a potent inhibitor of proliferation in others, such as lymphocytes and most epithelial cells. Moreover, it can regulate other processes that have little to do with cell division, such as the synthesis of collagen and other critical molecules of the extracellular matrix. Its actions are germane to almost every branch of medicine.
We briefly summarize the actions of TGF-ß in inflammation and repair of soft tissue, in formation of bone and cartilage, and in control of the immune system. In addition, we indicate its therapeutic potential in theses areas and discuss disease states in which the normal physiology of TGF-ß might be perturbed. Finally, we mention recent advances in the understanding of the role of TGF-ß in the heart and in carcinogeneses. Details can be obtained in two recent reviews.
Modern studies with peptide growth factors provide molecular confirmation of the old idea that repair of tissue injury represents a recapitulation of embryonic processes. Immunohistochemical and in situ hybridiazation studies have shown recently that TGF-ß is an important morphogenetic substance in the mammalian, embryo, particularly during the remodeling that occurs during formation of vertebrae, limb buds, teeth, facial bones, and the valves of the heart. High levels of TGF-ß normally are found in embryonic structures that are potential sites of congenital malformation, such as the palate or the interventricular septum of the heart. The formation and remodeling of connective tissue that occur during normal embryogeneses involve biochemical mechanisms similar to those used during repair of injury in the adult, and it seems that TGF-ß is an important mediator of both processes.
Inflammation and Repair
There is now extensive literature on the role of TGF-ß during inflammation and repair. Platelets are the most concentrated source of TGF-ß in the body, and TGF-ß is released from their alpha granules at sites of tissue injury. This process starts a repair cascade. Transforming growth factor-ß is the most potent known chemotactic factor for macrophages, being active at the picogram (10¯¹²) level. In addition to attracting macrophages to sites of tissue injury, TGF-ß activates their synthesis of other growth factors and deactivates their production of hydrogen peroxide, which would destroy nascent fibroblasts. Transforming growth factor-ß also has important chemotactic and anabolic actions on fibroblasts involved in tissue repair. It stimulates their production of critical components of extra-cellular matrix, such as collagen and fibronectin (as well as their receptors, known as "integrins") and proteoglycans, and it inhibits the action of proteolytic enzymes that destroy newly formed connective tissue. The net result of theses actions is the formation of new granulation tissue at the site of action of TGF-ß. This can be seen experimentally in animals injected subcutaneously with TGF-ß; a lump of highly vascularized granulation tissue forms rapidly (within 24 to 48 hours) at the injection site and disappears gradually when injections are stopped. Transforming growth factor-ß has been shown to enhance gene transcription of collagen, and this molecule plays a critical role in providing the structural strength of healing woulds as well as serving as an essential part of the matrix of bone and cartilage.
These actions are of obvious relevance for many potential clinical applications, including surgical wound healing in debilited patients or those undergoing chemotherapy, treatment of diabetic, decubitus, and varicose ulcers, and treatment of burns. Studies have shown that TGF-ß can enhance wound healing in animals, but human use has not begun yet. An important new application has been the intraocular administraion of TGF-ß after retinal reattachment in the underlying choroid. Clinical studies are planned.
There are disease states in which excessive production of TGF-ß or abnormal sensitivity of target cells to its actions may contribute to the pathogenesis of fibrosis, as has been suggested from clinical or animal studies on pulmonary fibrosis, hepatic cirrhosis, scleroderma, keloids, proliferative vitreoretinopathy, and rheumatoid arthritis. In the latter two diseases, significantly elevated levels of TGF-ß correlated with severity of disease.
Bone and Cartilage
At present there is an intense interest in the role of various peptide growth factors in the formation of cartilage and bone, with the goal of using such agents to enhance the repair of injured tissue or to arrest the process of osteoporosis. Chondrogenesis and osteogenesis are complex, multistep processes that involve several cell types and numerous peptide mediators that interact with each other in intricate feedback loops. It is likely that any effective future clinical use of peptide growth factors to promote formation of bone or cartilage may involve combined or sequential use of multiple factors that act as a set.
TGF-ß is one of the critical peptide growth factors that acts on chondrocytes or osteocytes. It is involved in the embryonic formation of cartilage and bone and is present in the growth plates of long bones. Large amounts also are found in adult bone; indeed, the second form of this peptide, TGF-ß2, was isolated originally from adult bovine bone.
Transforming growth factor-ß is a potent inducer of type II collagen and proteoglycans, which form the extracellular matrix of cartilage.
The physiological role of TGF-ß regulates the strict coupling of the processes of bone resorption and new bone formation that is characterisitic of the continuous remodeling that occurs in normal adult bone and that the actions of vitamin D and parathyroid hormone in remodeling might in some way be mediated by their effects on TGF-ß. The action of extradiol to increase bone matrix may be mediated by the ability of the estrogen receptor to activate the gene for TGF-ß, which in turn would promote formation of new bone matrix.
Studies are currently in progress in many laboratories to determine if TGF-ß can be used to accelerate fracture healing, since it acts both as a mitogen for osteoblasts as well as a stimulator of their production of extracellular matrix. Increased levels of messenger RNA for TGF-ß, as well as the peptide itself, have been found in the callus of healing fractures (M. Bolander, MD, S. Jingushi, MD, and M. Joyce, MD, oral communciation, May 1989). Moreover, injection of TGF-ß increases bone formation in vivo in rats. Two new proteins, know as "bone morphogenetic proteins," have been characterized and cloned and have been identified as more distant members of the extended TGF-ß gene family. It has been proposed to use bone morphogenetic proteins to enhance bone formation, and it will be important to determine whether they act synergistically with TGF-ß.
Little is known about the role of TGF-ß in the pathogenesis of bone disease. Given the new data on the role of estrogen in stimulating TGF-ß, further studies on its role in osteoporosis seem to be warranted. A related problem is the use of tamoxifen as a preventive or adjuvant agent for the treatment of breast cancer. Although tamoxifen often is considered to be an "antiestrogen," many of its actions seem to be independent of the estrogen receptor, and it has been shown that it maintains, rather than decreases, bone mass in extended clinical trials. Tamoxifen is a potent stimulator of TGF-ß secretion, and it is possible that a maintenance of bone mass may be mediated by this action. The above results all suggest that the develpoment of new pharmacologic agents that enhance TGF-ß formation in bone may provide a new therapeutic approach to osteoporosis.
Suppression of Lymphocite Function
TGF-ß is the most potent known endogenous suppressant of lymphocyte proliferation and function; it is 10 000 to 100 000 times more potent (on a molar basis) than cyclosporine. It is an endogenous product of both T and B cells, serving as an autocrine "stop" signal by inhibiting the action of the interleukins and other cytokines (such as tumor necrosis factor) that stimulate lymphocyte function. Thus, TGF-ß inhibits proliferaion of T cells stimulated by interleukin 1 or 2, inhibits proliferation and antibody production in B cells stimulated by any of several activating factors, depresses cytolytic activity of natural killer cells, and inhibits the generation of cytotoxic T cells and lymphokine-activated killer cells. Clearly, the immune systems needs its own brake to arrest excessive proliferation and activity when stimulated by antigens, and it seems that this is a pricipal role for TGF-ß.
The above effects have been confirmed amply in numerous in vitro studies and now form the basis of current attempts to use TGF-ß in vivo as an immunosuppressive agent. These studies are now in their earliest stages, but advances have been made, most notably in the suppression of cariac allograft rejection across major histocompatibility barriers in mice. Systemic administration of recombinant TGF-ß to recipient mice has improved acceptance of grafted hearts significantly (R. Morris, MD, and M. Palladino, PhD, oral communication, June 1989). The use of TGF-ß in experimental transplantation of allografts of liver, pancreatic islets, and kidneys also is under intensive study. Another area of current investigation is the use of TGF-ß to suppress inflammatory processes driven by activated T cells, as occurs in various types of experimental arthritis as well as in clinical rheumatoid disease.
Clinical data already suggest that high levels of TGF-ß are immunosuppressive. The immunosuppressed status of certain patients with cancer, most notably those with glioblastomas, may be the result of secretion of high levels of TGF-ß by their tumors; when these malignancies were removed surgically, more normal immune function returned. Indeed, human TGF-ß 2 was first isolated from a glioblastoma cell line and was characterized initially by its ability to suppress T-cell function. The possibility that exogenous immunosuppressive agents such as cyclosporine or dexamethasone might stimulate TGF-ß synthesis in lymphocytes currently is under investigation. Some of the toxic manifestations of cyclosporine, particularly its ability to induce renal fibrosis, also may be related to an induction of TGF-ß.
TGF-ß in the Heart
Immunohistochemical studies in rats and mice recently have shown an unusually large amount of intracellular TGF-ß in cardiac myocytes, cardiac vagal ganglia, and the conducting cells of the atrioventricular node. The physiological role of TGF-ß in normal cardiac function is unknown. We recently studied TGF-ß1 in the rat heart during myocardial infarction caused by ligation of the left coronary artery. Progressive loss of TGF-ß in myocytes occurred within 1 hour of coronary ligation, but 24 to 48 hours later, increased amounts were seen in myocytes at the margin of infarcted areas. Analysis of the TGF-ß1 messenger RNA in infarcted hearts showed the appearance of a new molecular species (a 1.9 kilobase transcript) in addition to a marked increase in the usual 2.4-kilobase transcript found in most tissues. The significance of these alterations in TGF-ß in the infarcted heart is unknown, and further studies are required to define its role in the pathogenesis and repair of cardiac injury. The possibility that exogenous TGF-ß delivered to a site of cardiac injury might accelerate repair now needs to be considered, especially in view of the known ability to facilitate other types of wound healing. As evidence accumulates that alteration in the extracellular collagen matrix of the heart are associated with pathological processes such as hypertrophy and ischemia, it will be important to study the role of TGF-ß in these conditions.
TGF-ß and Carcinogenesis
Although epithelial tissues constitute only a small percentage of the total weight of the body, most human cancers arise there. While epithelia are capable of rapid proliferation when injured, in many of them, such as those of the bronchi or urinary bladder, there is almost no cell division in the normal adult. The mechanisms responsible for restraint of proliferation in normal epithelia are only partially understood, but abundant data indicate that TGF-ß is a potent antiproliferative agent for most epithelial cells, including those from the mammary gland, liver, bronchus, kidney, skin, and intestine.
During the process of carcinogenesis epithelial cells, whose proliferation normally is suppressed by TGF-ß, may escape from autocrine or paracrine growth control by TGF-ß and become autonomous. The mechanisms involved are not well understood but can involve a loss of TGF-ß itself, a loss of the TGF-ß receptor on the putative tumor cell, or a failure in the intracellular growth control pathway mediated by TGF-ß. Any mechanism that result in the loss of such negative growth control may be associated with an enhanced susceptibility to malignancy. Development of pharmacologic agents that can enhance TGF-ß secretion in premalignant epithelia, before they have lost their sensitivity to TGF-ß, offers a new approach to chemoprevention of malignancy. As noted previously herein, tamoxifen is one such agent, and new data suggest that retinoic acid and its analogues (retinoids) also may act by a mechanism that involves TGF-ß (A. Glick, PhD, and S. Yuspa, MD, oral communication, June 1989). Both tamoxifen and retinoids have been used successfully to prevent either breast or skin cancer in experimental animals and are in current or planned clinical trials for prevention of malignancy in patients at high risk.
JAMA, August 18.1989 - Vol 262. No. 7