Insulin-Like Growth Factor I Is a Determinant of Hip Bone Mineral Density in Men Less Than 60 years of Age: MINOS Study

Several studies show that in elderly men bone mineral density (BMD) is not correlated with the insulin-like growth factor (IGF-I) level, but data are scanty in young men. Results of studies correlating insulin-like growth factor binding protein 3 (IGFBP-3) and BMD in men are discordant. As different hypotheses can explain the discordant results, we evaluated the correlation of BMD with serum IGF-I, IGFBP-3, and IGF-I/IGFBP-3 index in a large cohort of 721 men aged 19–85 years taking into account age, body weight, 17-estradiol, free testosterone, and parathyroid hormone. Serum IGF-I and IGFBP-3 decreased with age (r = –0.44 and r = –0.36, P = 0.0001). After adjustment for confounding variables, IGF-I correlated weakly positively with BMD and with bone mineral apparent density (BMAD) of hip as well as with cortical thickness of femoral neck, both of which are determined mainly by bone resorption, but not with bone size determined by periosteal apposition. IGF-I correlated weakly positively with BMD at the whole body and at the third lumbar vertebra IGFBP-3 and IGF-I/IGFBP-3 index did not correlate with densitometric parameters. In men aged 19–60 years, IGF-I correlated with BMD and BMAD of total hip and with cortical thickness of femoral neck positively and more strongly than in the entire cohort but not with the size of proximal femur. BMD of total hip was 6% higher in men in the highest quartile of IGF-I than in men in the lowest quartile. IGF-I, IGFBP-3, and IGF-I/IGFBP-3 index did not correlate with densitometric parameters of other sites. In the men aged more than 60 years, neither IGF-I nor IGFBP-3 nor IGF-I/IGFBP-3 index correlated with BMD, BMAD, or bone size. In men aged 19–60 years, the most significant hormonal determinants of BMD and BMAD of the hip and of the cortical thickness of femoral neck were 17-estradiol and IGF-I (P < 0.05–0.0001). In men aged more than 60 years, the most significant determinants of hip BMD were 17-estradiol and PTH. In conclusion, IGF-I seems to contribute to the inhibition of bone resorption and to maintaining bone mass of the proximal femur during the phase of slow bone loss in men aged less than 60 years. IGFBP-3 and IGF-I/IGFBP-3 index were not correlated with BMD or bone size. Supported by a contract with INSERM/Merck Sharp & Dohme Chibret, Paris, France     

Porcine osteochondrosis: Deficiencies in transforming growth factor-β and insulin-like growth factor-I

Osteochondrosis and dyschondroplasia are common multifocal disturbances of endochondral ossification in many species of domestic animals, and are characterized by the retention of avascular cartilage. These cartilage disorders are characterized by a failure of chondrocyte differentiation, matrix mineralisation and its replacement by bone. Rabbit polyclonal antibodies to transforming growth factor-beta (TGF-) and to insulin-like growth factor-I (IGF-I) were used to detect the two growth factors in normal and osteo-chondrotic porcine epiphyses. In the normal pig epiphyses IGF-I and TGF- were present in the chondrocytes of the epiphyseal hyaline cartilage and IGF-I was readily localised to the hypertrophic chondrocytes in the growth cartilage adjacent to the epiphyseal ossification centre. Both growth factors were found to be deficient in chondrocytes at sites of osteochondrosis. Both these growth factors are thought to be involved in the cascade of events associated with chondrocyte function during endochondral ossification. Deficiencies in TGF- and IGF-I demonstrated in porcine osteochondrosis and previously shown in avian dyschondroplasia suggest further similarities in the pathogenesis of these conditions.     

Potentiation of transforming growth factor (TGF-β1) by natural coral and fibrin in a rabbit cranioplasty model

The association of a biodegradable material and a growth factor could be of clinical value for treating bone defects. We therefore tested the association of transforming growth factor (TGF-1) in fibrin glue and coral granules to heal skull defects in rabbits. Adult rabbits underwent a double trepanation symmetrically in both parietal bones. Using histomorphometry, we compared bone repair after 1 month in control animals (n=5) and in animals treated with either TGF-1 as a single injection of 1 g in methylcellulose (n=5) or in fibrin glue (n=5), or with coral granules in fibrin glue (n=4) or with coral granules and TGF-1 1 g in fibrin glue (n=5). We measured the diameter of the remaining defect and the surface of the bone growth. TGF-1 without coral in either methyl cellulose or fibrin induced a partial closure of the defect as assessed by a significant decrease in the defect diameter, compared with the control group. However, the association of TGF-1 in fibrin and coral induced an area of the bone growth higher than in any other groups (P<0.05). Two months after surgery, this triple association induced a better healing of the defect than coral alone or control group. In each group treated with TGF-1, the mineralization rate was increased not only at the treated side but also in the contralateral defect which was untreated, suggesting a diffusion of the growth factor. Indeed, when pooled together, the diameter of the defect at the contralateral side of 14 animals that had received TGF-1 was reduced compared with the control group. Significant coral granules resorption occurred between month 1 and 2 and was unchanged by the addition of TGF-1. In conclusion, the triple association of coral granules and TGF-1 in fibrin could be of interest for treating bone defects.     

Demonstration of TGF-β1 mRNA by in situ hybridization in normal human fracture healing

Summary The role of transforming growth factor (TGF-) in fracture healing has previously been investigated in a rodent model, but not in human material. We investigated TGF-1 gene expression in specimens of callus from normally healing human fractures, using in situ hybridization to a cDNA TGF-1 probe and an autoradiographic disclosure system. TGF-1 mRNA was present in areas of proliferation of mesenchymal tissue, bone, and cartilage. Levels of expression were lower in cells in the fracture hematoma and in differentiated (hypertrophic) chondrocytes. These results are compatible with those found in various animal models using immunohistochemistry and support the view that locally produced TGF-1 is a regulator of fracture repair in humans from the early (mesenchymal proliferation) stage to the stage of remodeling of woven bone. They also indicate that, for TGF-1, animal models accurately reflect human bone repair.     

Transforming growth factor-beta localization during mouse prostate morphogenesis and in prostatic growth abnormalities

Summary Growth and morphogenesis of the prostate involves mesenchymal-epithelial interactions. Transforming growth factor-beta 1 (TGF-1) is one growth factor that may play a role in these paracrine interactions. We have localized TGF-1 by molecular and immunohistochemical analysis in the developing mouse prostate. Accumulations of TGF-1 protein were localized in the mesenchyme surrounding ductules in fetal and neonatal prostate. Previous studies in the mouse prostate reconstitution (MPR) model system have localized accumulations of TGF-1 to regions of oncogene-induced abnormalities. In surgically excised adult human prostate tissues, localized accumulations of TGF-1 are associated with prostate cancer and benign prostatic hyperplasia (BPH). Intracellular TGF-1 was more often associated with stromal cells in BPH and with neoplastic epithelial cells in prostate cancer. The production and accumulation of TGF-1 appears to involve interactions between mesenchymal and epithelial cells. Further experimental studies may clarify the relationships between TGF-1 and abnormal prostatic growth.     

Mechanical loading stimulates rapid changes in periosteal gene expression

Although mechanical forces regulate bone mass and morphology, little is known about the signals involved in that regulation. External force application increases periosteal bone formation by increasing surface activation and formation rate. In this study, the early tibial periosteal response to external loads was compared between loaded and nonloaded contralateral tibia by examining the results of blot hybridization analyses of total RNA. To study the impact of external load on gene expression, RNA blots were sequentially hybridized to cDNAs encoding the protooncogene c-fos, cytoskeletal protein -actin, bone matrix proteins alkaline phosphatase (ALP), osteopontin (Op), and osteocalcin (Oc), and growth factors insulin-like growth factor I (IGF-I) and transforming growth factor- (TGF-). The rapid yet transient increase in levels of c-fos mRNA seen within 2 hours after load application indirectly suggests that the initial periosteal response to mechanical loading is cell proliferation. This is also supported by the concomitant decline in levels of mRNAs encoding bone matrix proteins ALP, Op, and Oc, which are typically produced by mature osteoblasts. Another early periosteal response to mechanical load appeared to be the rapid induction of growth factor synthesis as TGF- and IGF-I mRNA levels were increased in the loaded limb with peak levels being observed 4 hours after loading. These data indicate that the acute periosteal response to external mechanical loading was a change in the pattern of gene expression which may signal cell proliferation. The altered pattern of gene expression observed in the present study supports previous evidence of increased periosteal cell proliferation seen both in vivo and in vitro following mechanical loading.Portions of this paper have been published in abstract form: J Bone Miner Res 7:S281, 1992     

ACE inhibition modulates transforming growth factor-β receptors in the young rat

The renin-angiotensin system plays an important role in renal growth and development. Exposure of the neonate to angiotensin converting enzyme (ACE) inhibitors increases mortality and results in growth retardation and abnormal renal development. It has been demonstrated that ACE inhibition in the developing kidney reduces the renal expression of growth factors, which may account for renal growth impairment. This study was designed to investigate the relationship between renal growth impairment and the expression of transforming growth factor-1 (TGF-1), TGF- receptor I [TRI, activin-like kinase (ALK)-1 and ALK-5], and TGF- receptor II (TRII). Newborn rat pups were treated with enalapril (30 mg/kg per day) or vehicle for 7 days, and kidneys were removed for Western blotting of TGF-1, ALK-1, ALK-5, and TRII, and for RT-PCR of ALK-5 and TRII. TGF-1, ALK-1, ALK-5, and TRII were also detected by immunohistochemistry. Enalapril treatment resulted in an increased mortality (30.4%) by day 7, and reduced body weight and kidney weight (P<0.05 versus vehicle). Enalapril decreased renal TGF-1, ALK-1, and ALK-5 protein expression (P<0.05). Also, enalapril decreased ALK-5 mRNA expression (P<0.05). TRII expression was not changed by enalapril treatment. These results indicate that ACE inhibition in the developing kidney decreases TGF-1, ALK-1, and ALK-5 expression, which may account for renal growth impairment. TRII may not be modulated by ACE inhibition in the developing kidney.     

Transforming Growth Factor-β1 Gene Polymorphisms and Bone Turnover,Bone Mineral Density and Fracture Risk in Southern Chinese Women

Genetic contributions play an important role in determining bone mineral density (BMD) and bone turnover. Transforming growth factor- (TGF-) is abundant in bone and has been implicated as an important regulator of both bone formation and resorption. Several polymorphisms of the TGF-1 gene have recently been suggested to be associated with BMD and susceptibility to osteoporotic spine fractures. To determine the relationship between TGF-1 polymorphisms and BMD in southern Chinese women, three SNPs at C^–1348-T, T²⁹-C, and T^861-20-C of TGF-1 gene were analyzed in 237 postmenopausal southern Chinese women by RFLP and direct sequencing. BMD at the lumbar spine and hip region, biochemical markers of bone turnover, as well as serum levels of TGF-1 were measured. Only the T²⁹-C polymorphism of TGF-1 gene was associated with BMD and fracture risk. The prevalence of fragility fractures was significantly higher in individuals with TC genotype (P < 0.05). Serum alkaline phosphatase and osteocalcin levels as well as urinary N-telopeptide excretion were significantly higher in women with TC than with TT or CC genotypes, and the difference remained significant after adjusting for age and BMI (all P < 0.05). Women with TC genotype had lower BMD at the trochanteric (P < 0.03) and total hip region (P = 0.05). No difference was observed in the serum TGF-1 levels among the three genotypes. In conclusion, an association between T²⁹-C polymorphisms of TGF-1 gene and BMD, bone turnover as well as fragility fractures were demonstrated in postmenopausal southern Chinese women.     

TGF-β and functional differentiation

A review of the pertinent literature suggests that TGF-1 may play a multifaceted role in functional differentiation of mammary epithelium. Evidence for the expression of TGF-1 RNA and the presence of functional TGF-1 protein in differentiating mammary epithelial cells from a pregnant mouse has been recently reported. The specific role of mammary-epithelial-cell-produced TGF-1 in the differentiating mammary gland is presently unclear. However, several possible functions are suggested from the following observations. Milk protein production is negatively regulated by exogenous TGF-1 during gestational development of the gland but not during lactation. Consistent with reports linking TGF-1 gene expression with mammary gland involution following lactation, overexpression of TGF-1 in the differentiating secretory epithelium leads to premature programmed cell death in the absence of a negative effect on secretory epithelial cell proliferation. A role for TGF-1 in cell cycle control and suppression of malignant progression independent from its inhibitory effect on epithelial cell growth has been demonstrated in keratinocytes. A similar function could provide protection against malignancy in proliferating mammary epithelium and account for TGF-1 suppression of mammary tumorigenesis in transgenic mice overexpressing transforming growth factor alpha (TGF-).³     

Effects of transforming growth factor-β1 on formation and activation of osteoclasts

Transforming growth factor-₁ (TGF-₁) has biological functions in various types of cells. However, its roles in the regulation of osteoclast formation and function are unclear. To examine them, we employed a culture system in which unfractionated cells obtained from long bones of 13-day-old mice were cultured on a dentine slice. We found that TGF-₁ has a potent inhibitory effect on osteoclastic bone resorption at a dose of 0.2–5 ng/ml. By electron microscopy the osteoclasts appeared to have fewer mitochondria and ruffled borders than those in control cultures. But in the presence of 1,25-dihydroxyvitamin D₃, [1,25-(OH)₂D₃], TGF-₁ at a dose of 0.2–1 ng/ml stimulated the formation of osteoclasts from unfractionated bone cell cultures in which preexistent osteoclasts had degenerated. Thus, using stromal cell-free he-mopoietic blast cells, we examined the direct action of TGF-₁ on osteoclast precursors. Although TGF-₁ inhibited tartrate-resistant acid phosphatase-positive (TRAP) multinucleate cell (MNC) formation induced by 1,25-(OH)₂D₃, the conditioned medium (CM) of TGF-₁-treated MC3T3-E1 cells stimulated such formation. These results suggest that TGF-₁ inhibits osteoclastic bone resorption but stimulates osteoclast formation via the action of factor(s) produced by TGF-₁-treated osteoblasts in the presence of 1,25-(OH)₂D₃.     

Quantification,Localization, and Expression of IGF-I and TGF-β1 During Growth Factor-Stimulated Fracture Healing

Because of the increasing interest on stimulating fracture healing, knowledge about the role and chronology of growth factors during the healing process is important. The purpose of this study was to quantify the protein concentration of IGF-I and TGF-1 during rat tibial fracture healing 5, 10, and 15 days after fracture using ELISA methods and to analyze the distribution of the proteins and the related mRNA expression in the fracture callus by immunohistochemistry and in situ hybridization. The following three groups were analyzed: Fractured tibiae intramedullary stabilized with K-wires coated with IGF-I and TGF-1 compared with fractures stabilized with uncoated K-wires and unfractured tibiae. The weight of the callus increased during the healing process in both experimental groups. The protein concentration of IGF-I and TGF-1 in the fracture callus showed significant changes between the investigated time points and treatment groups compared with the unfractured tibia. IGF-I increased with healing time whereas TGF-1 revealed a constantly elevated level at the investigated time points. Mesenchymal cells, osteoblasts, osteocytes, proliferating and immature chondrocytes, and osteoclasts expressed both growth factors. No differences in the expression and localization pattern of the growth factors were detectable among the groups. Using the different methods for quantification and visualization of the growth factors, no differences (except the increased IGF-I concentration at day 15 in the growth factor group) were seen between the normal and the growth factor-stimulated fracture healing as an indication for physiological healing after exogenous growth factor treatment. Parts of this study have been presented at the annual meeting of the ORS 2002 (Orthopaedic Research Society, Dallas, Feb. 2002) (Trans Orthop Res Soc 48^th, poster).     

Expression of Transforming Growth Factor Betas and Their Signaling Receptors in Stone-containing Intrahepatic Bile Ducts and Cholangiocarcinoma

Transforming growth factor betas (TGF-s) are multifunctional polypeptides that either inhibit or stimulate cell proliferation. They mediate their functions through their signaling receptors. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aim of this study was to examine the expression of TGF-s and their receptors in stone-containing intrahepatic bile ducts (IHD) and cholangiocarcinoma and try to predict whether hepatolithiasis has a predisposition to development of cholangiocarcinoma. Twenty-eight surgically resected specimens of stone-containing IHD and 15 specimens of cholangiocarcinoma were subjects for this study. Immunohistochemical analysis was done on three TGF-s and their signaling receptors to check their expression in non-neoplastic and neoplastic bile ducts. No immunoreactivity of TGF-₁ was found in any specimens. The overexpression of TGF-₂ and TGF-₃ was found in both hepatolithiasis (93%–100%) and cholangiocarcinoma (80%) at levels significantly higher than those of normal controls (10%–20%) (p < 0.001). The immunoreactivity of type I receptor (TRI) and type II receptor (TRII) also showed increased expression in stone-containing IHD, whereas TRII was absent in cholangiocarcinoma. We conclude that the overexpression of TGF-₂ and TGF-₃ and the absence of TRII in cholangiocarcinoma could lead to enhanced tumor cell proliferation. At the same time, the overexpression of TGF-s and their receptors in stone-containing IHD could suggest a close relationship between hepatolithiasis and cholangiocarcinoma.     

Latency and activation in the control of TGF-β

The biological activity of the transforming growth factor-'s (TGF-)³ is tightly controlled by their persistance in the extracellular compartment as latent complexes. Each of the three mammalian isoform genes encodes a product that is cleaved intracellularly to form two polypeptides, each of which dimerizes. Mature TGF-, a 24 kD homodimer, is noncovalently associated with the 80 kD latency-associated peptide (LAP). LAP is a fundamental component of TGF- that is required for its efficient secretion, prevents it from binding to ubiquitous cell surface receptors, and maintains its availability in a large extracellular reservoir that is readily accessed by activation. This latent TGF- complex (LTGF-) is secreted by all cells and is abundant both in circulating forms and bound to the extracellular matrix. Activation describes the collective events leading to the release of TGF-. Despite the importance of TGF- regulation of growth and differentiation in physiological and malignant tissue processes, remarkably little is known about the mechanisms of activationin situ. Recent studies of irradiated mammary gland reveal certain features of TGF-1 activation that may shed light on its regulation and potential roles in the normal and neoplastic mammary gland.     

Complex role of tumor cell transforming growth factor (TGF)-βs on breast carcinoma progression

Growth inhibition by the TGF-s has been extensively studied in both normal and transformed mammary epithelial cells. It has been proposed that loss of autocrine TGF- mediated growth regulation is a critical event in breast tumorigenesis and several lines ofin vitro andin vivo data support this hypothesis. However, a positive association between the expression of TGF-s by tumor cells and the progression or maintenance of breast cancinoma cells has been observed in many studies inin vivo tumor models. Possible mechanisms for these growth enhancing effects of TGF- include immunosuppression mediated by tumor TGF-s, enhanced angiogenesis, increased peritumoral stroma formation, and cell adhesion. The net effect of tumor cell TGF- on the biology of breast carcinogenesis would depend on the balance between autocrine growth inhibition of mammary epithelial cells and these growth enhancing effects.     

Differential expression of transforming growth factor-β1 and β3 as well as C-FOS mRNA in normal human prostate,benign prostatic hyperplasia and prostatic cancer

Summary The discrepancy between the incidence of latent prostate cancer and that of clinically overt carcinoma suggests that there can be different courses in the biological progression of prostate cancer. As this cancer is detected increasingly at an infraclinical stage, markers are needed to indicate which lesions will progress and lead to the patient's death. To investigate the possibility that specific growth factors and/or proto-oncogenes are expressed differentially, we measured mRNA levels of transforming growth factors 1 (TGF-1), TGF-2 and TGF-3 and of the c-fos and c-jun oncogenes by Northern blotting in normal prostate, benign prostatic hyperplasia (BPH) and prostate cancer. Our data demonstrate that expression of TGF-1 increased, whereas that of TGF-3 fell to an almost undetectable level in carcinoma. Expression of c-fos followed the TGF-1 pattern, whereas no difference could be seen in c-jun expression in cancer as compared with BPH and normal prostate. The differential expression of TGF-1, TGF-3 and c-fos could possibly be used to improve the characterisation of prostate cancer. Long-term follow-up of patients may indicate whether mRNA levels of these growth factors and oncogenes correlate clinically and whether they can be used as markers for progression in human prostate cancer.     

Differences in the Fusion and Resorption Activity of Human Osteoclasts After Stimulation with Different Growth Factors Released From a Polylactide Carrier

Previous in vivo studies were able to demonstrate the efficacy of locally released growth factors IGF-I, TGF-beta1, and BMP-2 from a poly(D,L-lactide) (PDLLA) implant coating on fracture healing. In vitro studies using human osteoblast-like cells showed an enhanced collagen-1 production due to growth factor application without an effect of the PDLLA on the investigated parameter. Both bone-forming osteoblasts and bone-resorbing osteoclasts are important during bone formation and fracture healing. The aim of this study was to investigate the influence of different growth factors and the polylactide coating into which they were incorporated on isolated osteoclasts. In vitro studies using human osteoclast-like cells derived from peripheral blood mononuclear cells (PBMNCs) were performed. Titanium K-wires coated with the lactide loaded with IGF-I and TGF-1 (alone and in combination) or BMP-2 were added to the culture in a non-contact manner and the fusion, resorption activity (pit formation assay), and TRAP 5b synthesis of the cells were analyzed. Differences in the effect of the growth factors were seen depending on the differentiation state of the cells. The fusion of the monocytes to multinuclear osteoclasts was significantly enhanced by the application of TGF-1 both alone and in combination with IGF-I. No effect was seen after application of IGF-I alone or BMP-2. The resorption activity of the osteoclasts analyzed on dentine chips was significantly enhanced after application of TGF-1 or BMP-2. These results indicate a differentiation-dependent effect of growth factors on osteoclasts. TGF-1 affects both the osteoclastogenesis and the activity of osteoclasts, whereas BMP-2 had an effect only on the activity of mature osteoclasts but not on the fusion of the PBMNCs.     

Transforming growth factor β-like activity in human hydrocele fluid

Summary In this study transforming growth factor (TGF-)-like activity in human hydrocele fluid was investigated. Inhibition of DNA synthesis of adult rat hepatocytes in primary culture and stimulation of colony formation of normal rat kidney (NRK) fibroblasts, clone 49F in soft agar were observed in all acidified hydrocele fluids and these activites were neutralized by the specific antibody raised against human native TGF-. In samples obtained from recurrent cases of hydrocele, TGF-like activity was observed in its active form (without acidification). These results suggest that human hydrocele fluid contains TGF-like activity and that the active form of TGF- in recurrent hydrocele fluid may be responsible for the recurrence of the disease even after repeated aspiration.     

Defects of TGF-β receptor signaling in mammary cell tumorigenesis

Transforming growth factor (TGF-)⁴ receptor expression and signal transduction in human breast cancer are reviewed as a function of estrogen receptor (ER) expression. ER⁺ breast cancer cells are generally resistant to the inhibitory effects of TGF-. The only known exception appears to be MCF-7 early passage cells which are initially sensitive to TGF-, but gain resistance after long-term passage in tissue culture. A number of studies have shown that loss of sensitivity is due to inadequate TGF- type II (TGFRII) receptor expression. Stable transfection of TGFRII into ER⁺ breast cancer cell lines results in the acquisition of TGF- sensitivity and reversion of malignancy. Although there are exceptions, ER^– breast cancer cells usually express TGFRII, but nevertheless show a low level of sensitivity to TGF-. Thus resistance in these cells implies a postreceptor mechanism. Given the frequency with which loss of TGF- sensitivity has been associated with loss of TGFRII, the ER^– breast cancer cell lines may represent valuable models for identifying postreceptor mechanisms of resistance.     

Growth factors and kidney development

The formation of the metanephric kidney is dependent upon the timed and sequential expression of a number of polypeptide growth factors. To shed light on the participation of members of the insulin-like growth factor (IGF) and epidermal growth factor/transforming growth factor- (EGF/TGF-) families, we measured the synthesis of IGF-I, IGF-II, EGF and TGF- by developing rat metanephroi in organ culture and determined the effect of anti-growth factor antibodies on growth and development. IGF-I, IGF-II and TGF- were produced by metanephroi and released into culture media. We could detect no EGF. Inclusion of anti-IGF-I, anti-IGF-II, anti-IGF-II receptor or anti-TGF- antibodies in organ cultures inhibited growth and development of metanephroi. Our findings suggest that both members of the IGF family and TGF- are produced within the developing metanephros and promote renal organogenesis.     

The role of TGF-β in patterning and growth of the mammary ductal tree

Evidence that transforming growth factor beta (TGF-)⁴ influences pattern formation in the developing mammary gland and negatively regulates ductal growth is reviewed. In the mouse, overexpression of TGF- transgenes during puberty reduces the rate of growth of the ductal tree and simplifies the pattern of arborization, while expression during pregnancy also interferes with lactation. Expression studies in the normal mouse gland indicate that TGF- is synthesized in the mammary epithelium, with the three isoforms showing somewhat different spatial and temporal distributions. Exogenous TGF- applied directly to the glandin situ inhibits epithelial cell division within hours, and strongly stimulates extracellular matrix synthesis over a longer time course. Normal human breast cells as well as certain breast cancer cell lines also secrete TGF- and are themselves inhibited by it, suggesting an autoregulatory feedback circuit, that in some cases appears to be modulated by estradiol. Taken together, the evidence suggests a model in which growth and patterning of the mammary ductal tree are regulated, at least in part, by TGF- operating through an autocrine feedback mechanism and by paracrine circuits associated with epithelial-stromal interactions.