Transforming Growth Factor-beta1 Gene Research Articles

Promoter sequences involved in transforming growth factor beta1 gene induction in HaCaT keratinocytes after gamma irradiation.

Transforming growth factor beta 1 (TGFB1) is a cytokine involved in the development of both acute and late cutaneous radiation syndromes. We previously demonstrated that ionizing radiation induces TGFB1 expression in vivo in pig skin within a few hours. The purpose of the present study was to develop an in vitro human model to identify the mechanisms of this early activation. Accordingly, human HaCaT keratinocytes were irradiated with a single dose of 20 Gy. First, radiation-induced TGFB1 overexpression was checked at both the transcriptional and transductional levels in HaCaT cells. Then electrophoretic mobility shift assays (EMSA) and transient transfection with various TGFB1 promoter constructs were used to identify the sequences involved in regulating this promoter. EMSA analysis showed the induction of nuclear protein binding activity by gamma irradiation to the -365 AP1 sequence (TGTCTCA), suggesting the involvement of AP1 sequences in the regulation of TGFB1 transcription. In gene reporter assays, maximal TGFB1 promoter activation was found for the longest construct, which contains two AP1 sequences. However, assays with constructs including deletions showed that these two AP1 sequences were not sufficient to confer TGFB1 inducibility. These results showed for the first time, to our knowledge, that transcriptional regulation is involved in radiation-induced activation of TGFB1 gene expression.

Production and purification of recombinant human inhibin and activin.

Inhibin and activin are protein hormones with diverse physiological roles including the regulation of pituitary FSH secretion. Like other members of the transforming growth factor-beta gene family, they undergo processing from larger precursor molecules as well as assembly into functional dimers. Isolation of inhibin and activin from natural sources can only produce limited quantities of bioactive protein. To purify large-scale quantities of recombinant human inhibin and activin, we have utilized stably transfected cell lines in self-contained bioreactors to produce protein. These cells produce approximately 200 microg/ml per day total recombinant human inhibin. Conditioned cell media can be purified through column chromatography resulting in dimeric mature 32-34 kDa inhibin A and 28 kDa activin A. The purified recombinant proteins maintain their biological activity as measured by traditional in vitro assays including the regulation of FSH in rat anterior pituitary cultures and the regulation of promoter activity of the activin-responsive promoter p3TP-luc in tissue culture cells. These proteins will be valuable for future analysis of inhibin and activin function and have been distributed to the US National Hormone and Peptide Program.

Association Between the T29->C Polymorphism in the Transforming Growth Factor beta1 Gene and Breast Cancer Among Elderly White Women: The Study of Osteoporotic Fractures--Correction

Association Between the T29->C Polymorphism in the Transforming Growth Factor beta1 Gene and Breast Cancer Among Elderly White Women: The Study of Osteoporotic Fractures--Correction

Cytokine polymorphic analyses indicate ethnic differences in the allelic distribution of interleukin-2 and interleukin-6.

Polymorphisms in the regulatory regions of cytokine genes affect protein production and are associated with allograft outcome. Ethnic origin has been identified as a significant prognostic factor for several immune-mediated diseases and for outcome after allotransplantation. A clear relationship between cytokine polymorphisms and ethnicity has not been shown. One hundred sixty subjects including 102 whites and 43 African-Americans were studied. Using polymerase chain reaction-based assays and, in some cases, restriction enzyme digestion, we determined genetic polymorphisms for the cytokines interleukin (IL) -2, IL-6, IL-10, tumor necrosis factor-alpha, transforming growth factor-beta, and interferon-gamma (IFN-gamma). Genetic polymorphism frequencies were then compared to ethnicity using chi-square analysis and Fisher's exact two-tailed tests. For both the IL-2 and IL-6 genes, we found that whites and African-Americans differed significantly (P <0.05) in their allelic distribution and genotype frequency. A trend toward ethnic distribution was noted among the alleles and genotypes for the IL-10 and IFN-gamma genes. We found no correlation between ethnicity and either allelic distribution or genotype frequency for the tumor necrosis factor-alpha or transforming growth factor-beta genes. When comparisons were made between patients with or without a history of kidney failure, the allelic or genotypic distributions for the IL-6 and IFN-gamma genes were found to significantly differ. Our work demonstrates a correlation between ethnicity and polymorphisms in several cytokine genes. In addition, we found that patients requiring renal transplantation differ from the general population with regard to certain cytokine gene polymorphisms. These findings may have relevance in making prognostic determinations or tailoring immunomodulatory regimens after renal transplantation.

Polymorphisms of angiotensinogen and transforming growth factor beta1 genes and disease progression in chronic hepatitis C

Polymorphisms of angiotensinogen and transforming growth factor beta1 genes and disease progression in chronic hepatitis C

Polymorphisms of angiotensinogen and transforming growth factor beta1 genes and disease progression in chronic hepatitis C

Polymorphisms of angiotensinogen and transforming growth factor beta1 genes and disease progression in chronic hepatitis C

Association of the C-509-->T polymorphism, alone of in combination with the T869-->C polymorphism, of the transforming growth factor-beta1 gene with bone mineral density and genetic susceptibility to osteoporosis in Japanese women.

Transforming growth factor-beta1 is an important local regulator of bone metabolism, acting downstream of estrogen and cooperatively with vitamin D. The possible association of a C 509-->T polymorphism in the promoter region of the transforming growth factor-beta1 gene, alone or in combination with a T869-->C (Leu10-->Pro) polymorphism, with bone mineral density and genetic susceptibility to osteoporosis was investigated in 625 postmenopausal Japanese women. The frequencies of the CC, CT, and TT genotypes of the C-509-->T polymorphism in the study population were 24%, 49%, and 27%, respectively. A significant association of C-509-->T genotype with bone mineral density was detected: lumbar spine (L2-L4) and total body bone mineral density values were 7% and 5% lower, respectively, in individuals with the TT genotype than in those with the CT or CC genotype. The serum concentration of transforming growth factor-beta1 did not vary with C-509-->T genotype. Multivariable logistic regression analysis, with adjustment for age, height, body weight, time since menopause, smoking status, body fat mass, and lean mass, revealed a significantly higher frequency of the TT genotype of the C-509-->T polymorphism in 286 individuals with osteoporosis than in 170 normal controls. Analysis of combined C-509-->T and T869-->C genotypes showed that L2-L4 bone mineral density decreases and the prevalence of osteoporosis increases with the number of T alleles. These results suggest that the C-509-->T polymorphism, alone or in combination with the T869-->C polymorphism, of the transforming growth factor-beta1 gene is a genetic determinant of bone mass, and that the number of T alleles in the combined genotype is a risk factor for the genetic susceptibility to osteoporosis in postmenopausal Japanese women.

Evidence of association and linkage disequilibrium between a novel polymorphism in the transforming growth factor beta 1 gene and hip bone mineral density: a study of female twins.

Bone mineral density (BMD) in later life is a major determinant of osteoporotic fracture risk and has been shown to be under strong genetic influence. Transforming growth factor beta 1 (TGF-beta 1) is an important regulatory cytokine, is found in high concentrations in the bone matrix, and is a plausible candidate for the genetic regulation of BMD. This study investigated whether a novel polymorphism within the TGF-beta 1 gene is associated with BMD in a large normal female population of 1706 dizygotic (DZ) twins (age range 18-76 yr). A C--->T [corrected] polymorphism was identified in intron 5, the T [corrected] allele having a frequency of 0.25. Subjects homozygous for the presence of the TGF-beta 1 T [corrected] allele had a 4% reduction in femoral neck BMD compared with the other two genotype groups (P<0.025). No effect was seen at the lumbar spine or ultradistal radius, or with calcaneal ultrasound measurements. Results were unaffected after adjustment for potential confounders. These findings were predominantly seen in pre-menopausal subjects, suggesting that this locus has an effect on the attainment of peak BMD. In pre-menopausal women, subjects who were homozygous for the T [corrected] allele had a 5-fold excess risk of having osteoporosis at the femoral neck compared with the other genotype groups. A within-pair analysis using the sibling transmission disequilibrium test confirmed these findings in pre-menopausal women and supported the candidacy of the TGF-beta 1 locus in the genetic regulation of hip BMD. These results indicate that allelic variation at the TGF-beta 1 gene contributes to the development of osteoporosis at the hip. The study also highlights the power of candidate gene analysis in twins, in whom loci having modest effects on disease risk can be identified.

Lack of Skin Fibrosis in Tight Skin (TSK) Mice with Targeted Mutation in the Interleukin-4Rα and Transforming Growth Factor-β Genes

Scleroderma is a disorder characterized by fibrosis of the skin and internal organs and autoimmunity. Whereas the cause is unknown, interleukin-4 and transforming growth factor-beta have been postulated to play a major part in the fibrosis. To investigate the part played by these cytokines, we prepared TSK/+ mice with a targeted mutation in the interleukin-4R alpha or transforming growth factor-beta genes. The breeding failed to produce TSK/+ transforming growth factor-beta -/- mice so analysis of the role of transforming growth factor-beta was limited to TSK/+ transforming growth factor-beta +/- mice. We observed that TSK/+ interleukin-4R alpha -/- did not develop dermal thickening, and deletion of one allele of the transforming growth factor-beta gene resulted in diminished dermal thickness compared with TSK/+ mice; however, the deletion of interleukin-4R alpha or transforming growth factor-beta had no effect on lung emphysema, which is another characteristic of TSK syndrome. Electron microscopic analysis of skin showed that the collagen fibrils in TSK/+ interleukin-4R alpha -/- mice exhibit normal periodicity but have a smaller diameter than the fibers found in C57BL/6 mice. Analysis of skin and serum samples showed that the deletion of interleukin-4R alpha or one allele of transforming growth factor-beta prevented the increase of skin thickness paralleled with a decrease in the dermal hydroxyproline content and development of autoantibodies associated with TSK syndrome. These results demonstrate the importance of interleukin-4 and transforming growth factor-beta for the development of cutaneous fibrosis in vivo and suggest an important part for these cytokines in wound healing and connective tissue maintenance in general.

Extracellular collagen regulates expression of transforming growth factor-beta1 gene.

A complex interrelationship exists between the extracellular matrix and cytokine signaling in articular chondrocytes. We sought to determine whether the extracellular matrix serves as a regulatory component of transforming growth factor-beta1 expression. Bovine articular chondrocytes were isolated and resuspended in alginate, yielding final extracellular protein concentrations of 0 to 1.5% (wt/vol) for type-II or type-I collagen. Cultures were maintained for 7 days in the presence or absence of transforming growth factor-beta1-supplemented medium (10 ng/ml). The amount of transforming growth factor-beta1 mRNA was examined with quantitative competitive reverse transcription-polymerase chain reaction analysis. The results indicate that exogenous transforming growth factor-beta1 stimulates endogenous transforming growth factor-beta1 mRNA expression approximately 8-fold. This effect depended on the concentration of extracellular type-II collagen. As the concentration of extracellular type-II collagen is increased, the expression of transforming growth factor-beta1 mRNA decreases in both basal and transforming growth factor-beta1-stimulated cultures. Exogenous extracellular type-I collagen also served to negatively modulate transforming growth factor-beta1 gene expression but with a different concentration profile. The results demonstrate that transforming growth factor-beta1 mRNA expression was upregulated by exogenous transforming growth factor-beta1 and was downregulated by extracellular type-I and type-II collagens. The profoundly different effects on transforming growth factor-beta1 expression by the two collagens are consistent with those reported for mammary epithelial cells and likely serve as a negative feedback mechanism to preserve tissue homeostasis.

Modulation of transforming growth factor beta-1 gene expression by interleukin-12.

The interaction of cytokines and their net balance with regard to macrophage activation (or deactivation) and immune stimulation (or suppression), ultimately determines the success of host-immune response at sites of active infection. A regulatory role for interleukin (IL)-12 in production of transforming growth factor (TGF)-beta1 has been suggested, however, remains controversial. In this study, we analyzed the effect of IL-12 on TGF-beta1 expression in the human lines, K562 and A549, and in primary human monocytes and macrophages. We found that IL-12 down-regulates TGF-beta1 mRNA expression in K562, monocytes and bone marrow cells, and to a lesser extent in the A549 cells. Using constructs containing different regions of the first promoter of the TGF-beta1 gene and a reporter gene, we also demonstrate that this effect is mediated through the TGF-beta1 gene promoter in the K562 and monocytic cell types. In conclusion, the critical role of IL-12 in the early activation of the immune response to pathogens may include down-modulation of TGF-beta1 gene activity.

Association of a polymorphism of the transforming growth factor beta-1 gene with prevalent vertebral fractures in Japanese women

Association of a polymorphism of the transforming growth factor beta-1 gene with prevalent vertebral fractures in Japanese women

Increased matrix synthesis following adenoviral transfer of a transforming growth factor beta1 gene into articular chondrocytes.

Monolayer cultures of lapine articular chondrocytes were transduced with first-generation adenoviral vectors carrying lacZ or transforming growth factor beta1 genes under the transcriptional control of the human cytomegalovirus early promoter. High concentrations of transforming growth factor beta1 were produced by chondrocytes following transfer of the transforming growth factor beta1 gene but not the lacZ gene. Transduced chondrocytes responded to the elevated endogenous production of transforming growth factor beta1 by increasing their synthesis of proteoglycan, collagen, and noncollagenous proteins in a dose-dependent fashion. The increases in collagen synthesis were not accompanied by alterations in the collagen phenotype; type-II collagen remained the predominant collagen. Transforming growth factor beta1 could not, however, rescue the collagen phenotype of cells that had undergone phenotypic modulation as a result of serial passaging. These data demonstrate that chondrocytes can be genetically manipulated to produce and respond to the potentially therapeutic cytokine transforming growth factor beta1. This technology has a number of experimental and therapeutic applications, including those related to the study and treatment of arthritis and cartilage repair.

Association of a T29-->C polymorphism of the transforming growth factor-beta1 gene with genetic susceptibility to myocardial infarction in Japanese.

Transforming growth factor-beta (TGF-beta) is an important regulator of vascular remodeling and is involved in the pathogenesis of atherosclerosis. A T-->C transition at nucleotide 29 of the TGF-beta1 gene results in a Leu-->Pro substitution at amino acid 10 of the signal peptide. We have now examined a possible association of TGF-beta1 genotype with myocardial infarction (MI) in a Japanese population. TGF-beta1 genotype was determined in 315 Japanese patients (234 men and 81 women) with MI and 591 control subjects (289 men and 302 women). We found that age, body mass index, and incidence of habitual smoking, hypertension, diabetes mellitus, and hypercholesterolemia did not differ between the 2 groups for either men or women. Multivariable logistic regression analysis, however, demonstrated the frequency of the T allele to be significantly higher in male subjects with MI than in controls (TT + TC versus CC; P<0.0001, odds ratio 3.5, 95% CI 2.0 to 6.3). In contrast, the T allele was not associated with the prevalence of MI in women. In both male MI patients and controls, the serum concentration of TGF-beta1 was significantly higher in individuals with the CC genotype than in subjects with the TT or TC genotype. Findings suggest that the T allele at nucleotide 29 in the TGF-beta1 gene is a risk factor for genetic susceptibility to MI, at least in middle-aged Japanese men.

Incisional wound healing in transforming growth factor-beta1 null mice.

Expression of endogenous transforming growth factor-beta1 is reduced in many animal models of impaired wound healing, and addition of exogenous transforming growth factor-beta has been shown to improve healing. To test the hypothesis that endogenous transforming growth factor-beta1 is essential for normal wound repair, we have studied wound healing in mice in which the transforming growth factor-beta1 gene has been deleted by homologous recombination. No perceptible differences were observed in wounds made in 3-10-day-old neonatal transforming growth factor-beta1 null mice compared to wild-type littermates. To preclude interference from maternally transferred transforming growth factor-beta1, cutaneous wounds were also made on the backs of 30-day-old transforming growth factor-beta1 null and littermate control mice treated with rapamycin, which extends their lifetime and suppresses the inflammatory response characteristic of the transforming growth factor-beta1 null mice. Again, no impairment in healing was seen in transforming growth factor-beta1 null mice. Instead these wounds showed an overall reduction in the amount of granulation tissue and an increased rate of epithelialization compared to littermate controls. Our data suggest that release of transforming growth factor-beta1 from degranulating platelets or secretion by infiltrating macrophages and fibroblasts is not critical to initiation or progression of tissue repair and that endogenous transforming growth factor-beta1 may actually function to increase inflammation and retard wound closure.

Transforming growth factor-beta1 gene and protein expression associated with atherogenesis of cholesterol-fed rabbits.

Transforming growth factor-beta1 (TGF-1beta) has been shown to modulate both cell proliferation and the synthesis of extracellular matrix by vascular cells. This study was aimed to establish the temporal correlation between TGF-beta1 expression, the expression of the extracellular matrix protein fibronectin, and plaque development during atherogenesis of hypercholesterolemic rabbits. New Zealand White rabbits were fed with 2% cholesterol-supplemented chow for 1 week, 2 weeks, 3 weeks or 6 weeks. TGF-beta1 mRNA and protein expression was examined in serial sections of aorta by in situ hybridization and immunohistochemistry. Fibronectin expression was examined by immunohistochemistry. In the control and 1-week feeding group, the expression of TGF-beta1 mRNA and protein was not apparent. In 2-week feeding group, intimal thickening was detected in which TGF-beta1 mRNA and protein were not clearly observed, either. The 3-week and 6-week feeding groups exhibited fatty streaks in which TGF-beta1 mRNA and protein expression markedly increased as feeding proceeded. Cell type-specific staining indicated that TGF-beta1 was expressed by macrophages as well as smooth muscle cells of the fatty streaks. Immunostaining of fibronectin detected low expression levels in control, 1-week and 2-week feeding groups with pronounced upregulation in the thickened intima and the proximal media in 3-week and 6-week feeding groups. These results implicate a role for TGF-beta1 in modulating fatty streak formation and the synthesis of extracellular protein fibronectin during plaque development.

Modulation of transforming growth factor beta1 gene expression in the mammary gland by insulin-like growth factor I and octreotide.

Transforming growth factor beta1 (TGF-beta1) has been shown to exhibit anti-proliferative activity for mammary gland epithelial cells and for human breast cancer cells. Insulin-like growth factor I (IGF-I), in contrast, is a well-characterized mitogenic and anti-apoptotic factor involved in mammary gland physiology. In order to examine the hypothesis that IGF-I suppresses TGF-beta1 expression in the mammary gland, we studied the effect of various manipulations of the growth hormone - IGF-I axis on TGF-beta1 mRNA abundance. Administration of IGF-I to intact animal suppressed TGF-beta1 mRNA levels in a dose-dependent manner to approximately 20% of control levels. Administration of the somatostatin analogue octreotide in a manner previously shown to acutely suppress the growth hormone - IGF-I axis increased mammary gland TGF-beta1 expression approximately 3-fold. Transgenic mice overexpressing growth hormone expressed TGF-beta1 in the mammary gland at only approximately 12% of the level of control animals, while mice IGF-I deficient due to the mutation expressed TGF-beta1 at slightly higher levels than control animals. The large differences in TGF-beta1 expression between control and GH-transgenic animals were correlated with major differences in architecture of the mammary gland, while the appearance of mammary glands of normal and animals was similar. These data document a previously unrecognized relationship between TGF-beta1 and IGF-I physiology in the mammary gland, and suggest a novel mechanism by which somatostatin analogues influence the proliferative behaviour of breast epithelial cells.

Intragraft expression of transforming growth factor beta1 gene in isolated glomeruli from human renal transplants.

Experimental evidence suggests that transforming growth factor (TGF) beta1 is a fibrogenic cytokine. The histopathological changes of chronic renal allograft nephropathy are dominated by fibrotic changes and TGF-beta may have an important aetiological role. This study investigated the relationship between intragraft TGF-beta gene expression and extracellular matrix protein deposition in human renal allografts. Sixteen cadaveric renal transplant recipients immunosuppressed with cyclosporin and steroids were studied. Individual glomeruli were isolated from protocol needle-core biopsies and, following messenger RNA extraction, intragraft gene expression was studied by reverse transcriptase-polymerase chain reaction. Collagen III deposition in these renal transplant biopsies was examined by immunohistochemistry and quantified by computerized histomorphometry. There was a positive correlation between renal cortical collagen III immunostaining and the levels of glomerular complementary DNA for TGF-beta1. TGF-beta1 is a profibrotic influence in human renal transplants. The methods described should prove of benefit in investigating the mechanisms of chronic renal allograft damage.

Transforming growth factor beta-1 gene polymorphism and bone mineral density in japanese adolescents

Osteoporosis has a strong genetic component (1). Among the genes that may be associated with bone mineral density are those encoding the vitamin D (2,3) and estrogen (4) receptors, collagen Iα1 (5,6), and apolipoprotein E (7). Transforming growth factor β1 (TGF-β1), which is abundantly stored in bone, is an important regulator of skeletal development and homeostasis. TGF-β1 has been shown to stimulate the proliferation and differentiation of osteoblasts, as well as their production of matrix, and to enhance bone formation in vivo (8). In addition, TGF-β1 has been implicated as a local mediator of the skeletal effects of estrogen, such as the estrogen-induced apoptosis of osteoclasts, thereby protecting against excessive bone resorption (9).

Transcriptional Regulation of the Bmp2Gene: RETINOIC ACID INDUCTION IN F9 EMBRYONAL CARCINOMA CELLS ANDSACCHAROMYCES CEREVISIAE

Bmp2, a highly conserved member of the transforming growth factor-beta gene family, is crucial for normal development. Retinoic acid, combined with cAMP analogs, sharply induces the Bmp2 mRNA during the differentiation of F9 embryonal carcinoma cells into parietal endoderm. Retinoic acid (RA) also induces the Bmp2 gene in chick limb buds. Since normal Bmp2 expression may require an endogenous retinoid signal and aberrant Bmp2 expression may cause some aspects of RA-induced teratogenesis, we studied the mechanism underlying the induction of Bmp2. Measurements of the Bmp2 mRNA half-life and nuclear run-on assays indicated that RA stimulated the transcription rate of the Bmp2 gene. The results of ribonuclease protection and primer extension assays indicated that Bmp2 transcription started 2,127 nucleotides upstream of the translation start site in F9 cells. To identify genetic elements controlling this transcription rate increase, upstream and downstream genomic sequences flanking the Bmp2 gene were screened using chloramphenicol acetyltransferase reporter genes in F9 cells and beta-galactosidase reporter genes in Saccharomyces cerevisiae that were cotransformed with retinoic acid receptor and retinoid X receptor expression plasmids. RA-dependent transcriptional activation was detected between base pairs -2,373 and -2,316 relative to the translation start site. We also identified a required Sp1 binding site between -2,308 and -2,298. The data indicate that Bmp2 is directly regulated by retinoic acid-bound receptors and Sp1.