Growth Inhibitory Effects of Transforming Growth Factor-β1 in vivo

Abstract

Transforming growth factor-beta (TGF-β) can reversibly inhibit the in vitro proliferation of murine and human haemopoietic progenitors and. some of their more developmentally restricted progeny. Using an assay for measuring day 8 and day 11 CFU-S, TGF-β caused a gradual decline in the number of CFU-S undergoing DNA synthesis so that after 5 days of daily treatment only quiescent cells were found. Release of this growth inhibition was seen within 24 hours post-treatment with recovery of all progenitors to normal levels. Similar inhibitory effects of TGF-β were seen on the cells of the intestinal epithelium, indicating that TGF-β is a general stem cell growth inhibitor. These results suggest that TGF-β can be used as a cytostatic agent to protect normal stem cells in patients being treated with cell cycle-specific cytotoxic agents.     

Expression of Tumor Necrosis Factor-α and Transforming Growth Factor-β1 in Acute Liver Injury

Abstract

Tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) have a number of in vitro functions that could be important in vivo in acute liver injury and repair. Therefore, we investigated these two cytokines in acute liver damage. Northern blots of RNA isolated from rats sacrificed at various time intervals after a single oral dose of CCl₄ revealed that TNF-α mRNA levels were elevated within 6 hr of CCl₄ administration and returned to control values by 24–32 hr. In contrast, TGF-β1 mRNA levels started to rise significantly at 24 hr, peaked at 48 hr, and approached baseline levels by 72 hr. Identical changes in TNF-α and TGF-β1 mRNA levels were also seen with D-galactosamine-induced hepatotoxicity. Immunohistochemical analysis using a TGF-β1 antibody demonstrated increased hepatic staining in CCl₄-treated rats, at times corresponding to the increases in TGF-β1 gene expression. Therefore, there is a differential expression of these cytokines in acute CCl₄ and galactosamine hepatotoxicity with an early rise in TNF-α, suggesting that this cytokine may affect inflammation and cell toxicity, while TGF-β1 peaks later, when it may regulate hepatocyte proliferation and extracellular matrix repair.     

Localization of Cells Containing Transforming Growth Factor- α Precursor Immunoreactivity in the Basal Ganglia of the Adult Rat Brain

Abstract

Transforming growth factor-α-like immunoreactivity (TGF-α-ir) was visualized in the adult rat forebrain using three antisera directed against carboxyterminal sequences in the TGF-α precursor. Using immunoperoxidase and immunofluorescence techniques with all three antisera, TGF-λ-ir was found to be present in a subpopulation of astrocytes in the forebrain. Striatal and pallidal regions of the basal ganglia were studied in detail. In the striatum, there was an uneven distribution of astrocytes containing TGF-α-ir, with the greatest number in the dorsal medial third of the caudate-putamen and the overlying corpus callosum/external capsule. In addition, the region of the caudate-putamen bordering the globus pallidus contained numerous clusters of TGF-α-ir astrocytes. The globus pallidus itself contained numerous and more evenly distributed TGF-α-ir astrocytes. Other pallidal structures-including the ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata–contained moderate numbers of TGF-α-ir astrocytes. These results suggest that TGF-α precursor is present and, perhaps, synthesized in astrocytes. A related growth factor, epidermal growth factor (EGF), has also been reported to be present in pallidal regions of rat brain. Therefore, the TGF-α/EGF family of trophic factors may play an important role in the function of the central nervous system.     

Accelerated Healing of Ulcer Wounds in the Rabbit Ear by Recombinant Human Transforming Growth Factor-β1

Abstract

A dermal ulcer wound-healing model was established in rabbit ear to examine the effects of recombinant human transforming growth factor-β1 (rhTGF-β 1) in wound healing. Histo-morphometric examination of the wounds indicate a biphasic healing response 7 days after a single application of rhTGF-β l at the time of wounding. Statistically significant healing occurred at 5-100 ng but not at higher doses of 500 or 1000 ng rhTGF-β l/wound. Enhanced collagen synthesis as determined by [³H] proline incorporation occurred at 15 and 25 ng and was significantly depressed at 500 ng rhTGF-β1wound. Multiple doses of 100 ng rhTGF-/3l applied to the wound at the time of wounding and for 3 days after wounding provided results comparable to the single application of growth factor. Delaying treatment 24 hr after wounding did not enhance wound healing compared with vehicle. Our findings suggest that rhTGF-β l can be a valuable growth factor to improve the healing of ulcer wounds.     

Elevation of Transforming Growth Factor-β1 Is Associated with Recurrent Miscarriage

To investigate the significance of transforming growth factor-1 (TGF-1) in reproduction we have compared plasma levels in normal pregnant women and patients suffering miscarriages. We examined 188 normal pregnant women and 12 pregnant women with miscarriages. Eight women with severe recurrent miscarriages (mean ± SD of previous number of miscarriages; 10.4 ± 2.4 times) were also examined before conception; 34 nonpregnant women served as controls. Plasma TGF-1 level increased with the gestational week and returned within the normal range 1 month after delivery. The levels among pregnant women with miscarriages (mean ± SD; 2.44 ± 0.83 ng/ml) were significantly higher than those of pregnant controls (1.74 ± 0.95 ng/ml) of matched gestational weeks; levels among nonpregnant women with severe recurrent miscarriages were extremely elevated (4.1 ± 3.04 ng/ml) compared to the control value (1.34 ± 0.59 ng/ml). These data suggest that TGF-1 may be necessary to maintain pregnancy but also may be a risk factor for recurrent miscarriages.     

Downregulation of Decorin and Transforming Growth Factor-β1 by Decorin Gene Suppression in Tendinocytes

Scars formed after tendonitis result in altered tissue mechanical properties after injury. The interaction of collagen molecules with decorin affects collagen fibrogenesis, and scar tissue is fragile as a consequence of a large amount of decorin in the scar. We hypothesized that scar formation could be prevented by controlling decorin expression in tendinocytes. As a preliminary experiment, we treated tendinocytes with decorin antisense oligodeoxynucleotides (ODNs). Tendinocytes were isolated from Achilles tendons of New Zealand white rabbits and treated with ODN. When tendinocytes were transfected with decorin sense ODN, there was no alteration, whereas decorin antisense ODN-treated tendinocytes showed suppression of transforming growth factor (TGF)-β1 production. Decorin and TGF-β1-production of tendinocytes is regulated by decorin gene suppression. The results showed that the antisense approach is an attractive therapeutic strategy not only for preventing decorin deposition in scar tissue, which decreases collagen fibril diameter, but also for controlling TGF-β1 production, which leads to organ fibrosis.     

Influence of Transforming Growth Factor-��1 Gene Polymorphism at Codon 10 on the Development of Cirrhosis in Chronic Hepatitis B Virus Carriers

Transforming growth factor (TGF)-β1 is a key cytokine producing extracellular matrix. We evaluated the effect of TGF-β1 gene polymorphism at codon 10 on the development of cirrhosis in patients with chronic hepatitis B. One hundred seventy eight patients with chronic hepatitis (CH, n=57) or liver cirrhosis (LC, n=121), who had HBsAg and were over 50 yr old, were enrolled. The genotypes were determined by single strand conformation polymorphism. There were no significant differences in age and sex ratio between CH and LC groups. HBeAg positivity and detection rate of HBV DNA were higher in LC than in CH groups (P=0.055 and P=0.003, respectively). There were three types of TGF-β1 gene polymorphism at codon 10: proline homozygous (P/P), proline/leucine heterozygous (P/L), and leucine homozygous (L/L) genotype. In CH group, the proportions of P/P, P/L, and L/L genotype were 32%, 51%, and 17%, respectively. In LC group, the proportions of those genotypes were 20%, 47%, and 33%, respectively. The L/L genotype was presented more frequently in LC than in CH groups (P=0.017). Multivariate logistic regression analysis confirms that detectable HBV DNA (odds ratio [OR]: 3.037, 95% confidence interval [CI]: 1.504-6.133, P=0.002) and L/L genotype (OR: 3.408, 95% CI: 1.279-9.085, P=0.014) are risk factors for cirrhosis.     

Transforming Growth Factor-β–Independent Role of Connective Tissue Growth Factor in the Development of Liver Fibrosis

We previously identified transforming growth factor (TGF)-β signaling as a fibronectin-independent mechanism of type I collagen fibrillogenesis following adult liver injury. To address the contribution of TGF-β signaling during the development of liver fibrosis, we generated adult mice lacking TGF-β type II receptor (TGF-βIIR) from the liver. TGF-βIIR knockout livers indeed showed a dominant effect in reducing fibrosis, but fibrosis still remained approximately 45% compared with control and fibronectin knockout livers. Unexpectedly, this was accompanied by significant up-regulation of connective tissue growth factor mRNA levels. Organized type I collagen networks in TGF-βIIR knockout livers colocalized well with fibronectin. We provide evidence that elimination of TGF-βIIR is not sufficient to completely prevent liver fibrosis. Our results indicate a TGF-β–independent mechanism of type I collagen production and suggest connective tissue growth factor as its potent mediator. We advocate combined elimination of TGF-β signaling and connective tissue growth factor as a potential therapeutic target by which to attenuate liver fibrosis.Liver fibrosis is defined as an abnormal response to persistent liver injury, characterized by the excessive accumulation of collagenous extracellular matrices (ECMs).^{1, 2} Liver fibrosis affects tens of millions of people worldwide and is of great clinical significance because normal liver architecture is disrupted and liver function is ultimately impaired. Because there is no effective treatment of liver fibrosis, many patients develop progressive liver cirrhosis, eventually requiring a liver transplant.^{3, 4}There is a long-standing concept that cells in culture cannot form a collagen fibril network without the ECM glycoprotein fibronectin.⁵ We recently established an adult mouse model lacking liver fibronectin and demonstrated that fibronectin-null livers, in fact, formed collagen fibril networks similarly to wild-type mice in response to carbon tetrachloride–induced chronic liver injury. The networks were found to be nucleated by type V collagen, induced by elevated local transforming growth factor (TGF)-β bioavailability.⁶ Therefore, we identified two mechanisms of collagen fibrillogenesis in response to liver injury: both fibronectin and TGF-β–signaling mediated.Early in the fibrogenic process, inflammatory cytokines are important in initiating repair following injury. TGF-β acts as a fibrogenic master cytokine and plays a pivotal role in the progression of a variety of chronic fibrotic diseases by promoting myofibroblastic differentiation, stimulating synthesis of ECMs, and down-regulating ECM degradation.⁷ In TGF-β–mediated signaling, ligand TGF-βs bind to TGF-β type I and type II receptors that form heterotetrameric complexes. On ligand binding, downstream Smad signaling pathways are initiated. Activated (phosphorylated) Smads translocate to the nucleus where they are involved in the regulation of gene expression.^{8, 9} Currently, several monoclonal antibodies and small molecules targeting TGF-β are in the process of clinical application for chronic fibrotic diseases, including liver fibrosis.¹⁰ However, these studies were initiated without knowledge of how TGF-β signaling exerts its action in the development of liver fibrosis.Elevated TGF-β activity in chronic fibrotic diseases is often accompanied by elevated expression of a matricellular protein, connective tissue growth factor (CTGF/CCN2).¹¹ The manifestation of CTGF/CCN2 functions in vivo involves cooperative interactions with costimulatory factors in the microenvironment, such as TGF-β and fibronectin. One hypothesis arising from the in vivo models is that fibrosing liver injuries are exacerbated by the action of TGF-β–mediated CTGF/CCN2.¹² Here, we addressed the extent to which fibrosis is dependent on the TGF-β/CTGF/CCN2 axis in chronic liver injury using an adult mouse model lacking liver TGF-β type II receptors (TGF-βIIR).     

Modulation of Transforming Growth Factor-β Actions in Rat Osteoblast-like Cells: The Effects of bFGF and EGF

Abstract

The present study examines how the mitogenic and differentiation functions of transforming growth factor-β (TGF-β) are modulated by basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in primary cultures of rat osteoblast-like (ROB) cells. TGF-β, bFGF, and EGF individually stimulated [³H]thymidine incorporation and cell proliferation in a dose range of 0.01-10 ng/ml. When studied in combination, high doses of bFGF and EGF were additive to low doses of TGF-β. The additive effects of bFGF and EGF on mitogenesis diminished with increasing doses of TGF-β. These three factors also decreased alkaline phosphatase activity individually within the same dose range. When cells were treated with the combined factors, only high doses of bFGF and EGF were additive to the TGF-β inhibition. We were unable to detect any change in collagen synthesis with each individual factor or in combined treatments. In addition, TGF-β or bFGF alone or in combination did not affect fibronectin synthesis. Our studies showed that the biological functions of TGF-β can be modulated by bFGF and EGF in ROB cells. The pattern of modulation is varied depending on the specific function examined.     

Vitamin D3 and Calcipotriol Enhance the Secretion of Transforming Growth Factor-β1 and -β2 in Cultured Murine Keratinocytes

Abstract

Vitamin D₃ and its analogue calcipotriol (MC 903) inhibit the proliferation of cultured keratinocytes and induce their differentiation. Since TGFβs are very potent inhibitors of keratinocyte growth we studied the effects of vitamin D₃ and calcipotriol on the secretion of TGFβ in cultured murine keratinocytes. Vitamin D, and calcipotriol (10^?6 - 10^?9 M) inhibited the DNA-synthesis of mouse keratinocytes by 50–80% in a time and dose-dependent manner as measured by [³H]-thymidine incorporation. Analysis of the conditioned medium of the keratinocytes indicated that the cells secreted into their medium activity that inhibited the growth of indicator Mv1Lu mink lung epithelial cells. Neutralizing antibodies against TGFβ1 and TGFβ2 decreased, and when used together, prevented the observed growth inhibition of the indicator cells. Heat treatment of the conditioned medium, which activates latent forms of TGFβ, revealed higher levels of growth inhibitory activity in the medium from vitamin D₃ and calcipotriol treated than from control cultures indicating that a fraction of TGFβ was in a latent form. Active TGFβ was, however, detected considerably more in vitamin D₃ and calcipotriol treated cultures than in control cultures. Immunoblotting analysis of the medium revealed enhanced secretion of TGFβ protein. These results indicate that enhanced TGFβ1 and TGFβ2 secretion and activity is associated with vitamin D₃-mediated growth inhibition of cultured keratinocytes.

This work was presented in part at the Keystone Symposium “Negative Growth Control”, Keystone, CO, Jan. 26-Feb. 2, 1992 (Koli and Keski-Oja 1992).     

Suppressed Production of Transforming Growth Factor-β2 mRNA in Endometrium of Women with Polycystic Ovary Syndrome

Production of transforming growth factor-₂ mRNA in the endometrium of women with polycystic ovary syndrome decreased compared to normal and this decrease directly depends on the duration of anovulatory period (from 3 weeks to 4 months). Low production of transforming growth factor-₂ mRNA probably contributes to the development of endometrial hyperplasia in women with polycystic ovary syndrome.     

EZH2 Promotes Extracellular Matrix Degradation via Nuclear Factor-κB (NF-κB) and p38 Signaling Pathways in Pulpitis

Inflammation - Pulpitis is a complicated chronic inflammatory process which can be in a dynamic balance between damage and repair. The extracellular matrix plays an important regulatory role in...     

Transforming Growth Factor-β Supports the Rapid Morphogenesis of Heterotopic Endochondral Bone Initiated by Human Osteogenic Protein-1 via the Synergistic Upregulation of Molecular Markers

Members of the transforming growth factor-β (TGF-β) superfamily of proteins, the bone morphogenetic proteins (BMPs) and the TGF-β isoforms, are involved in the coordination of cartilage and bone differentiation both in embryonic development and in postnatal life. Both osteogenic protein-1 (OP-1) and TGF-β1 have been shown to be potent regulators and inducers of heterotopic endochondral bone induction in non-human primates. In marked contrast, TGF-β1 does not induce heterotopic endochondral bone in rodents. In the primate, the osteogenic properties of OP-1 are synergistically enhanced by the combined administration of TGF-β1. The binary application of OP-1 (0.1, 0.3, 1.0 and 3.0μg) and TGF-β1 (0.01, 0.03 and 0.1 μg) to 25 mg of guanidinium-inactivated insoluble collagenous bone matrix as carrier in the rodent heterotopic bioassay for 7, 12 and 21 days resulted in a classical synergistic, dose-dependent and temporal up-regulation of OP-1-induced endochondral bone formation. There were significant increases in alkaline phosphatase activity (day 12) and calcium content (days 12 and 21). mRNA expression of OP-1, TGF-β1, BMP-3 and collagens type II and IV, markers of bone formation, showed an up-regulation of the genes (days 12 and 21) by the binary applications of the morphogens. Histologically, single applications of OP-1 elicited a dose dependent induction of endochondral bone formation while the binary applications resulted in a temporal acceleration of the morphogenetic cascade. The optimal ratio of OP-1/TGF-β1 was 30:1 by weight for endochondral bone formation and expression of molecular markers. The present data provides insights to the mechanisms of synergistic molecular therapeutics for endochondral bone formation in clinical contexts.