Polymorphism in codons 10 and 25 of the transforming growth factor‐beta 1 (TGF‐β1) gene in patients with invasive squamous cell carcinoma of the uterine cervix

Transforming growth factor‐beta 1 (TGF‐β1) has a multifactorial role in the development of cervical cancer. It potently inhibits the growth of epithelial cells that harbour oncogenic human papilloma viruses (HPVs). TGF‐β1 also inhibits the expression of the early viral transforming regions E6 and E7, which appear to be the key oncoproteins. It has been suggested that squamous cell carcinomas are devoid of TGF‐β1, raising the possibility that elevated levels of this growth factor could protect against cervical cancer. It is also recognized that the production and levels of TGF‐β1 are genetically predetermined and individually variable. Two genetic polymorphisms in the DNA encoding the leader sequence of the TGF‐β1 gene have been described and shown to be associated with the production of high or low TGF‐β1 levels in vivo and in vitro. We hypothesized that the inheritance of these polymorphisms could influence the development of invasive cervical cancer. This hypothesis was investigated by studying polymorphism in codons 10 and 25 of the TGF‐β1 gene. We studied 97 patients with invasive cervical cancer and 73 healthy controls and found that the distributions of alleles T (Leu) and/or C (Pro) and alleles G (Arg) and/or C (Pro) in codons 10 and 25, respectively, were similar. There was no significant association between the alleles and the histological degree of cancer differentiation. It appears that the role of this growth factor in cervical oncogenesis is not related to the point mutations that we examined in codons 10 and 25 of the TGF‐β1 gene. We speculate that other factors, including additional polymorphisms of the TGF‐β1 gene, the status of TGF‐β1 receptors, the complex cytokine network, differential responsiveness of cells to the stimuli, and the status of the precancer/cancer genome, may play a role in development of invasive cervical cancer.     

Transforming Growth Factor‐β Suppressed Id‐1 Expression in a smad3‐Dependent Manner in LoVo Cells

TGF‐β plays an important role in regulating cell differentiation and proliferation in human cancers such as colorectal cancer. Id‐1 has been identified as a marker in colorectal cancer progression. The aim of this study was to investigate the role of TGF‐β in regulating Id‐1 in LoVo cells. siRNA was used to silence smad2, smad3, and p38 MAPK gene expression in Lovo cells. Interference efficiency and the role of TGF‐β on Id‐1 expression were analyzed using a luciferase reporter assay, RT‐PCR, and Western blotting. Cell viability was determined using the MTT assay. In this study, we demonstrated that TGF‐β1 downregulated Id‐1 protein expression in LoVo cells. Smad2 and smad3 siRNA inhibited TGF‐β1‐induced 4×SBE luciferase reporter activity. p38 MAPK siRNA inhibited TGF‐β1‐induced 3×AP‐1 luciferase reporter activity. However, the suppression of Id‐1 by TGF‐β1 was recovered by smad3 siRNA but not smad2 or p38 MAPK siRNA. Moreover, TGF‐β1 stimulated cellular proliferation and p21^Waf1 protein expression, which might be mediated by suppressing Id‐1 expression. In conclusion, this study demonstrated that TGF‐β1 suppressed Id‐1 expression in a smad3‐dependent manner in LoVo cells using RNAi technology. These results provide new insight into the mechanisms of TGF‐β function in colorectal cancer cells. Anat Rec, 2010. © 2009 Wiley‐Liss, Inc.     

CDKL5 deficiency predisposes neurons to cell death through the deregulation of SMAD3 signaling

CDKL5 deficiency disorder (CDD) is a rare encephalopathy characterized by early onset epilepsy and severe intellectual disability. CDD is caused by mutations in the X‐linked cyclin‐dependent kinase‐like 5 (CDKL5) gene, a member of a highly conserved family of serine‐threonine kinases. Only a few physiological substrates of CDKL5 are currently known, which hampers the discovery of therapeutic strategies for CDD. Here, we show that SMAD3, a primary mediator of TGF‐β action, is a direct phosphorylation target of CDKL5 and that CDKL5‐dependent phosphorylation promotes SMAD3 protein stability. Importantly, we found that restoration of the SMAD3 signaling through TGF‐β1 treatment normalized defective neuronal survival and maturation in Cdkl5 knockout (KO) neurons. Moreover, we demonstrate that Cdkl5 KO neurons are more vulnerable to neurotoxic/excitotoxic stimuli. In vivo treatment with TGF‐β1 prevents increased NMDA‐induced cell death in hippocampal neurons from Cdkl5 KO mice, suggesting an involvement of the SMAD3 signaling deregulation in the neuronal susceptibility to excitotoxic injury of Cdkl5 KO mice. Our finding reveals a new function for CDKL5 in maintaining neuronal survival that could have important implications for susceptibility to neurodegeneration in patients with CDD.     

Loss of caveolin‐1 in prostate cancer stroma correlates with reduced relapse‐free survival and is functionally relevant to tumour progression

Levels of caveolin‐1 (Cav‐1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav‐1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav‐1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav‐1 in the stroma correlated with reduced relapse‐free survival (p = 0.009), suggesting a role for stromal Cav‐1 in inhibiting advanced disease. Silencing of Cav‐1 by shRNA in WPMY‐1 prostate fibroblasts resulted in up‐regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5‐fold increase in TGF‐β1 and γ‐synuclein (SNCG) gene expression. Moreover, silencing of Cav‐1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down‐regulation of TGF‐β1 and SNCG, suggesting that loss of Cav‐1 in the stroma can influence Akt‐mediated signalling in the tumour microenvironment. Cav‐1‐depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav‐1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up‐regulation of TGF‐β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Three‐generation family with novel contiguous gene deletion on chromosome 2p22 associated with thoracic aortic aneurysm syndrome

Latent transforming growth factor binding proteins (LTBP) are a family of extracellular matrix glycoproteins that play an important role in the regulation of transforming growth factor beta (TGF‐ß) activation. Dysregulation of the TGF‐ß pathway has been implicated in the pathogenesis of inherited disorders predisposing to thoracic aortic aneurysms syndromes (TAAS) including Marfan syndrome (MFS; FBN1) and Loeys–Dietz syndrome (LDS; TGFBR1, TGFBR2, TGFB2, TGFB3, SMAD2, SMAD3). While these syndromes have distinct clinical criteria, they share clinical features including aortic root dilation and musculoskeletal findings. LTBP1 is a component of the TGF‐ß pathway that binds to fibrillin‐1 in the extracellular matrix rendering TGF‐ß inactive. We describe a three‐generation family case series with a heterozygous ～5.1 Mb novel contiguous gene deletion of chromosome 2p22.3‐p22.2 involving 11 genes, including LTBP1. The deletion has been identified in the proband, father and grandfather, who all have a phenotype consistent with a TAAS. Findings include thoracic aortic dilation, ptosis, malar hypoplasia, high arched palate, retrognathia, pes planus, hindfoot deformity, obstructive sleep apnea, and low truncal tone during childhood with joint laxity that progressed to reduced joint mobility over time. While the three affected individuals did not meet criteria for either MFS or LDS, they shared features of both. Although the deletion includes 11 genes, given the relationship between LTBP1, TGF‐ß, and fibrillin‐1, LTBP1 stands out as one of the possible candidate genes for the clinical syndrome observed in this family. More studies are necessary to evaluate the potential role of LTBP1 in the pathophysiology of TAAS.

     

Transforming growth factor‐β1 genotype in sporadic breast cancer patients from India: status of enhancer,promoter, 5′‐untranslated‐region and exon‐1 polymorphisms

Transforming growth factor beta (TGF‐β) is an example for a large and still‐growing family of growth factors. TGF‐β1 is known to act both as a tumour suppressor and as a stimulator of tumour progression. This study examines the relationship amongst putative enhancer, promoter, 5′‐untranslated‐region (UTR) and exon‐1 polymorphisms of the TGF‐β1 gene (region I from ?1881 to ?1613; region II from ?1410 to ?1123, and region III from ?55 to +176, as per human genome organisation (HUGO) nomenclature) in 26 breast cancer patients and 97 healthy control subjects. The germline and somatic status of the four known polymorphisms was ascertained, and a significant difference was observed for the germline C/T and T/T genotype distribution between patients and controls in comparison to C/C genotypes at position ?1349 (χ² = 6.193; P = 0.009). In addition to the somatic variations observed for some of the regions studied, in 10/26 (38%) sporadic breast cancer cases, a novel somatic mutation in codon 47 of exon 1 (GenBank accession number AY059373 ) was also detected in tumour samples. The risk of cancer was found to be significant (OR = 4.525) for the ?1349 C/T and T/T genotype background, suggesting that this genetic background may act as a risk factor for sporadic breast cancer.     

Deciphering TGF‐β3 function in medial edge epithelium specification and fusion during mouse secondary palate development

Background: Transforming growth factor‐β3 (TGF‐β3) plays a central role in mediating secondary palate fusion along the facial midline. However, the mechanisms by which TGF‐β3 functions during secondary palate fusion are still poorly understood. Results : We found that mouse cytokeratin 6α and 17 mRNAs were expressed exclusively in the palate medial edge epithelium on embryonic day 14.5, and this expression was completely abolished in Tgf‐β3 mutant embryos. In contrast, we found that Jagged2 was initially expressed throughout the palate epithelium, but was specifically down‐regulated in the medial edge epithelium during palatal fusion. Jagged2 down‐regulation was regulated by TGF‐β3, since Jagged2 was persistently expressed in palatal medial edge epithelium in Tgf‐β3 null mutant embryos. Moreover, addition of DAPT, a specific inhibitor of Notch signaling, partially rescued the fusion defects in Tgf‐β3 null mutant palatal shelves. Conclusions : Based on these results, together with the previous study indicating that the loss of Jagged2 function promotes embryonic oral epithelial fusion, we concluded that TGF‐β3 mediates palate fusion in part by down‐regulating Jagged2 expression in palatal medial edge epithelium. In addition, cytokeratin 6α and 17 are two TGF‐β3 downstream target genes in palate medial edge epithelium differentiation. Developmental Dynamics 243:1536–1543, 2014. © 2014 Wiley Periodicals, Inc.     

Transforming growth factor‐β/Smad ‐ signalling pathway and conjunctival remodelling in vernal keratoconjunctivitis

Background Vernal keratoconjunctivitis (VKC) is a chronic ocular allergic inflammation characterized by corneal complications and the formation of giant papillae. Sma‐ and Mad‐related proteins (Smad) modulate extracellular matrix gene expression during wound healing, inflammation and tissue remodelling. Objective To investigate the relationship between allergic inflammation and TGF‐β/Smad signalling pathway, expression in VKC patients and in primary cultured conjunctival fibroblasts exposed to mediators found previously over‐expressed in VKC. Methods Smad‐2, ‐3, ‐7, phospho‐(p)Smads, TGF‐β1 and ‐β2 were evaluated in the conjunctiva of normal subjects (CT) and VKC patients by immunohistochemistry. The expression of Smads, pro‐collagen I (PIP), TGF‐β1, ‐β2, mitogen‐activated protein kinase (p38/MAPK), c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK1/2) were also determined in conjunctival fibroblast cultures exposed to histamine, IL‐4, ‐13, TGF‐β1, IFN‐γ and TNF‐α using immunostaining or RT‐PCR. Results Immunostaining for Smad‐2, ‐3, pSmad‐2, ‐3, TGF‐β1, ‐β2 and PIP was significantly increased in VKC stroma compared with CT. In conjunctival fibroblast cultures, Smad‐3 and PIP were stimulated by histamine, IL‐4, ‐13 and TGF‐β1 exposure, while PIP was reduced by IFN‐γ, and TNF‐α mRNA expression of Smad‐3 was increased by histamine, while Smad‐7 was reduced by IL‐4. In addition, histamine, IL‐4 and TNF‐α increased JNK and ERK1/2 expression. Conclusion and Clinical Relevance The TGF‐β/Smad signalling pathway is over‐expressed in VKC tissues and modulated in conjunctival fibroblasts by histamine, IL‐4, TGF‐β1 and TNF‐α. These mechanisms may be involved in fibrillar collagen production, giant papillae formation and tissue remodelling typical of VKC and might provide new therapeutic targets for its treatment. Cite this as: A. Leonardi, A. Di Stefano, L. Motterle, B. Zavan, G. Abatangelo and P. Brun, Clinical & Experimental Allergy, 2011 (41) 52–60.     

Titration of non-replicating adenovirus as a vector for transducing active TGF-β1 gene expression causing inflammation and fibrogenesis in the lungs of C57BL/6 mice

Investigators have shown that interstitial pulmonary fibrosis (IPF) can be induced in rats by overexpressing transforming growth factor beta₁ (TGF‐β₁) through a replication‐deficient recombinant adenovirus vector instilled into the lungs ( Sime et al. 1997 ). We have shown that this vector induces IPF in fibrogenic‐resistant tumour necrosis factor alpha‐receptor knockout (TNF‐αRKO) mice ( Liu et al. 2001 ). The object of our studies is to understand how peptide growth factors, such as TGF‐β₁, mediate interstitial lung disease (ILD). To do so, we must be able to manipulate the dose of the factor and sort out its effects on multiple other mediators in the lung parenchyma. As a step in this complex process, in the studies reported here, we have determined the concentrations of the recombinant adenovirus vector carrying the gene for porcine active TGF‐β₁ (AVTGFβ₁) that have little apparent effect, cause clear induction of disease, or severe disease. The disease largely resolves by 28 days in all cases, thus providing a valuable model to understand the mechanisms of the IPF that is mediated, at least in part, by TGF‐β₁. The findings here show that 10⁶ plaque‐forming units (pfu) of AVTGFβ₁, provide essentially a ‘no‐effect’ dose, but even this amount of TGF‐β₁ causes a significant increase in whole‐lung collagen by day 28 after treatment. In contrast, 10⁸ and 10⁹ pfu cause severe IPF in 4 days, whereas 10⁷ and 5 × 10⁷ are intermediate for all parameters studied, i.e. TGF‐β protein, inflammatory cells, cell proliferation, pro‐α 1(I) collagen gene expression and whole‐lung collagen accumulation, and expression of growth factors such as TGF‐β₁, TNF‐α and PDGF‐A and ‐B. Interestingly enough, TGF‐β₁, as a potent blocker of epithelial cell proliferation, appears to suppress airway epithelial cell growth that would be expected during the inflammatory phase of IPF. Thus, this model system helps us to understand some quantitative aspects of TGF‐β₁ biological activity and allows us to manipulate this potent factor as a mediator of interstitial fibrogenesis.     

Loss of tumour suppressor PTEN expression in renal injury initiates SMAD3‐ and p53‐dependent fibrotic responses

Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)‐mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK‐2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI‐1, vimentin, α‐SMA and fibronectin expression, compared to HK‐2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53^Ser15 phosphorylation, with an accompanying decrease in population density and an increase in epithelial G₁ cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI‐1 rescued the PTEN loss‐associated epithelial proliferative arrest. Moreover, TGFβ1‐initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53‐dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3‐ and p53‐mediated fibrotic gene induction, with accompanying PAI‐1‐dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Transforming growth factor-β1 gene polymorphisms in Korean women with endometriosis

Citation Lee HJ, Kim H, Ku S‐Y, Kim SH, Kim JG. Transforming growth factor‐β1 gene polymorphisms in Korean women with endometriosis. Am J Reprod Immunol 2011; 66: 428–434 Problem To investigate the association between endometriosis, transforming growth factor‐β1 (TGFB1) gene polymorphisms, and serum TGF‐β1 levels in Korean women. Method of study The ?509C/T, 868T/C, 913G/C and 979G/A polymorphisms of the TGFB1 gene were analyzed in women with (n = 131) and without (n = 107) endometriosis using restriction fragment length polymorphism (RFLP) analysis. Serum TGF‐β1 levels were measured by enzyme‐linked immunosorbent assay (ELISA). Results The 913G/C and 979G/A polymorphisms were not observed in the study participants. The genotype and allele distribution of the ?509C/T and 868T/C polymorphisms in endometriosis were similar to those in controls. However, the ?509T/868C (TC) haplotype allele was observed 4.55 times more frequently in early‐stage endometriosis than in other haplotype alleles. Serum TGF‐β1 levels were significantly higher in endometriosis than in controls. The single and haplotype genotype of ?509C/T and 868T/C polymorphisms were not related with serum TGF‐β1 levels. Conclusion The TC haplotype allele of TGFB1?509C/T and 868T/C polymorphisms may be associated with early‐stage endometriosis in Korean women.     

Opposing effects of pro‐ and anti‐inflammatory cytokine gene polymorphisms on the risk for breast cancer in western Indian women: a pilot study

In an earlier study, the genotypes associated with higher level of transforming growth factor‐β1 (TGF‐β1) were found to reduce the risk for breast cancer in western Indian women. This observation implied that gene polymorphisms affecting the levels of pro‐ and anti‐inflammatory cytokines may influence the risk for breast cancer in this population. Hence, we performed genotyping for three more functional single‐nucleotide polymorphisms (SNPs) responsible for variations in the levels of cytokines associated with inflammation. To that effect, polymorphisms in genes coding for IL‐4 (IL‐4 C‐590T; rs2243250), IFN‐γ (IFN‐G A + 874T; rs2430561) and MCP‐1 (MCP‐1 A‐2578G; rs1024611) were examined in premenopausal, healthy women (N = 239) and patients with breast cancer (N = 182) from western India. In carriers of the IL‐4*590T allele, a reduced risk for the disease (dominant model; OR = 0.61, 95% CI 0.37–0.98) was seen similar to that seen in TGF‐B1*10C carriers. An opposite trend was observed with respect to the alleles associated with higher expression of MCP‐1 or IFN‐γ. In individuals positive for three or more alleles associated with higher levels of either pro‐ or anti‐inflammatory cytokines, an additive effect on the modulation of risk for the disease was evident (for TGF‐B1 & IL‐4, OR = 0.33, 95% CI 0.12–0.87; for IFN‐G & MCP‐1, OR = 2.29, 95% CI 0.95–5.51). In the context of contrasting observations in other populations, these results indicate a significant contribution of anti‐inflammatory genotypes in the modulation of risk for breast cancer in western Indian women.     

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF,IGF, TGF‐β, TNF,and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β₃, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999. © 1999 Wiley‐Liss, Inc.