Generation of mice carrying a knockout‐first and conditional‐ready allele of transforming growth factor beta2 gene

Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell‐matrix remodeling, epithelial‐mesenchymal transition, and wound healing in a highly context‐dependent and tissue‐specific manner. Tgfb2^?/? mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2^βgeo (knockout‐first lacZ‐tagged insertion) gene‐trap allele and Tgfb2^flox conditional allele. Tgfb2^βgeo/βgeo or Tgfb2^βgeo/‐ mice died at perinatal stage from the same congenital heart defects as Tgfb2^?/? mice. β‐galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2^βgeo fetal tissue sections. Tgfb2^flox mice were produced by crossing the Tgfb2^+/βgeo mice with the FLPeR mice. Tgfb2^flox/? mice were viable. Tgfb2 conditional knockout (Tgfb2^cko/?) fetuses were generated by crossing of Tgfb2^flox/? mice with Tgfb2^+/?; EIIaCre mice. Systemic Tgfb2^cko/? embryos developed cardiac defects which resembled the Tgfb2^βgeo/βgeo, Tgfb2^βgeo/?, and Tgfb2^?/? fetuses. In conclusion, Tgfb2^βgeo and Tgfb2^flox mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis 52:817–826, 2014. © 2014 Wiley Periodicals, Inc.     

Generation of mice with a conditional allele for the transforming growth factor beta3 gene

The transforming growth factor beta (TGFβ) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFβ3 (Tgfb3) encodes one of the three ligands for TGFβ receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3-/- mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3-/- mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFβ3 function in different cell or tissue types in embryonic development and during adulthood.     

Loss of tenascin X gene function impairs injury‐induced stromal angiogenesis in mouse corneas

To determine the contribution by tenascin X (Tnx) gene expression to corneal stromal angiogenesis, the effects were determined of its loss on this response in TNX knockout (KO) mice. In parallel, the effects of such a loss were evaluated on vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) gene and protein expression in fibroblasts and macrophages in cell culture. Histological, immunohistochemical and quantitative RT‐PCR changes determined if Tnx gene ablation on angiogenic gene expression, inflammatory cell infiltration and neovascularization induced by central corneal stromal cauterization. The role was determined of Tnx function in controlling VEGF‐A or TGFβ1 gene expression by comparing their expression levels in ocular fibroblasts and macrophages obtained from wild‐type (WT) and body‐wide Tnx KO mice. Tnx was up‐regulated in cauterized cornea. In Tnx KO, macrophage invasion was attenuated, VEGF‐A and its cognate receptor mRNA expression along with neovascularization were lessened in Tnx KOs relative to the changes occurring in their WT counterpart. Loss of Tnx instead up‐regulated in vivo mRNA expression of anti‐angiogenic VEGF‐B but not VEGF‐A. On the other hand, TGFβ1 mRNA expression declined in Tnx KO cultured ocular fibroblasts. Loss of Tnx gene expression caused VEGF‐A expression to decline in macrophages. Tnx gene expression contributes to promoting TGFβ1 mRNA expression in ocular fibroblasts and VEGF‐A in macrophages, macrophage invasion, up‐regulation of VEGF‐A expression and neovascularization in an injured corneal stroma. On the other hand, it suppresses anti‐angiogenic VEGF‐B mRNA expression in vivo.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

TGF‐β promotes cell death and suppresses lactation during the second stage of mammary involution

Transforming growth factor beta (TGF‐β) ligands are known to regulate virgin mammary development and contribute to initiation of post‐lactation involution. However, the role for TGF‐β during the second phase of mammary involution has not been addressed. Previously, we have used an MMTV‐Cre transgene to delete exon 2 from the Tgfbr2 gene in mammary epithelium, however we observed a gradual loss of TβRII deficient epithelial cells that precluded an accurate study of the role for TGF‐β signaling during involution timepoints. Therefore, in order to determine the role for TGF‐β during the second phase of mammary involution we have now targeted TβRII ablation within mammary epithelium using the WAP‐Cre transgene [TβRII^(WKO)Rosa26R]. Our results demonstrated that TGF‐β regulates commitment to cell death during the second phase of mammary involution. Importantly, at day 3 of mammary involution the Na–Pi type IIb co‐transporter (Npt2b), a selective marker for active lactation in luminal lobular alveolar epithelium, was completely silenced in the WAP‐Cre control and TβRII^(WKO)Rosa26R tissues. However, by day 7 of involution the TβRII^(WKO)Rosa26R tissues had distended lobular alveoli and regained a robust Npt2b signal that was detected at the apical luminal surface. The Npt2b abundance and localization positively correlated with elevated WAP mRNA expression, suggesting that the distended alveoli were the result of an active lactation program rather than residual milk protein and lipid accumulation. In summary, the results suggest that an epithelial cell response to TGF‐β signaling regulates commitment to cell death and suppression of lactation during the second phase of mammary involution. J. Cell. Physiol. 219: 57–68, 2009. © 2008 Wiley‐Liss, Inc.     

The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes

The transforming growth factor-beta 1 (TGFβ1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/− and −/− keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/− mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFβ1.     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Transforming growth factor‐β1 requires NADPH oxidase 4 for angiogenesis in vitro and in vivo

Angiogenesis, the formation of new blood vessels, is a key physiological event in organ development and tissue responses to hypoxia but is also involved in pathophysiologies such as tumour growth and retinopathies. Understanding the molecular mechanisms involved is important to design strategies for therapeutic intervention. One important regulator of angiogenesis is transforming growth factor‐β1 (TGF‐β1). In addition, reactive oxygen species (ROS) and the ROS‐forming NADPH oxidase type 4 (Nox4) have been implicated as additional regulators such as during hypoxia. Here, we show that both processes are indeed mechanistically linked. TGF‐β1‐stimulated Nox4 expression and ROS formation in endothelial cells. In cells from Nox4‐deficient mice, TGF‐β1‐induced cell proliferation, migration and tube formation were abolished. In vivo, TGF‐β1 stimulated growth of blood vessels into sponges implanted subcutaneously, and this angiogenesis was markedly reduced in Nox4 knockout mice. Thus, endothelial cells are regulated by a TGF‐β1 signalling pathway involving Nox4‐derived ROS to promote angiogenesis. In order to abrogate pathological angiogenesis triggered by a multitude of factors, such as TGF‐β1 and hypoxia, Nox4 may thus be an ideal therapeutic target.     

Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants

Although TGF-β isoforms (TGF-β1-3) display very similar biochemical characteristics in vitro, it has been determined that they demonstrate different or even opposing effects in vivo. During embryogenesis, TGF-βs play important roles in several developmental processes. Tgfb3 is strongly expressed in the prefusion palatal epithelium, and mice lacking Tgfb3 display a cleft of the secondary palate. To test whether the effect of TGF-β3 in palatogenesis is isoform-specific in vivo, we generated a knockin mouse by replacing the coding region of exon1 in the Tgfb3 gene with the full-length Tgfb1 cDNA, which resulted in the expression of Tgfb1 in the Tgfb3 expressing domain. The homozygote knockin mice display a complete fusion at the mid-portion of the secondary palate, while the most anterior and posterior regions fail to fuse appropriately indicating that in vivo replacement of TGF-β3 with TGF-β1 can only partially correct the epithelial fusion defect of Tgfb3 knockout embryos. Palatal shelves of Tgfb1 knockin homozygote mice adhere, intercalate, and form characteristic epithelial triangles. However, decreased apoptosis in the midline epithelium, slower breakdown of the basement membrane and a general delay in epithelial fusion were observed when compared to control littermates. These results demonstrate an isoform-specific role for TGF-β3 in the palatal epithelium during palate formation, which cannot be fully substituted with TGF-β1.     

Aggregatibacter actinomycetemcomitans outer membrane protein 29 (Omp29) induces TGF‐β‐regulated apoptosis signal in human gingival epithelial cells via fibronectin/integrinβ1/FAK cascade

Gingival junctional epithelial cell apoptosis caused by periodontopathic bacteria exacerbates periodontitis. This pathological apoptosis is involved in the activation of transforming growth factor β (TGF‐β). However, the molecular mechanisms by which microbes induce the activation of TGF‐β remain unclear. We previously reported that Aggregatibacter actinomycetemcomitans (Aa) activated TGF‐β receptor (TGF‐βR)/smad2 signalling to induce epithelial cell apoptosis, even though Aa cannot bind to TGF‐βR. Additionally, outer membrane protein 29 kDa (Omp29), a member of the Aa Omps family, can induce actin rearrangements via focal adhesion kinase (FAK) signalling, which also plays a role in the activation of TGF‐β by cooperating with integrin. Accordingly, we hypothesized that Omp29‐induced actin rearrangements via FAK activity would enhance the activation of TGF‐β, leading to gingival epithelial cell apoptosis in vitro. By using human gingival epithelial cell line OBA9, we found that Omp29 activated TGF‐βR/smad2 signalling and decreased active TGF‐β protein levels in the extracellular matrix (ECM) of cell culture, suggesting the transactivation of TGF‐βR. Inhibition of actin rearrangements by cytochalasin D or blebbistatin and knockdown of FAK or integrinβ1 expression by siRNA transfection attenuated TGF‐βR/smad2 signalling activity and reduction of TGF‐β levels in the ECM caused by Omp29. Furthermore, Omp29 bound to fibronectin (Fn) to induce its aggregation on integrinβ1, which is associated with TGF‐β signalling activity. All the chemical inhibitors and siRNAs tested blocked Omp29‐induced OBA9 cells apoptosis. These results suggest that Omp29 binds to Fn in order to facilitate Fn/integrinβ1/FAK signalling‐dependent TGF‐β release from the ECM, thereby inducing gingival epithelial cell apoptosis via TGF‐βR/smad2 pathway.     

TRAF6 promotes TGFβ-induced invasion and cell-cycle regulation via Lys63-linked polyubiquitination of Lys178 in TGFβ type I receptor

Transforming growth factor β (TGFβ) can act either as a tumor promoter or a tumor suppressor in a context-dependent manner. High levels of TGFβ are found in prostate cancer tissues and correlate with poor patient prognosis. We recently identified a novel TGFβ-regulated signaling cascade in which TGFβ type I receptor (TβRI) is activated by the E3 ligase TNF-receptor-associated factor 6 (TRAF6) via the Lys63-linked polyubiquitination of TβRI. TRAF6 also contributes to activation of TNF-α-converting enzyme and presenilin-1, resulting in the proteolytic cleavage of TβRI and releasing the intracellular domain of TβRI, which is translocated to the nucleus to promote tumor invasiveness. In this report, we provide evidence that Lys178 of TβRI is polyubiquitinated by TRAF6. Moreover, our data suggest that TRAF6-mediated Lys63-linked ubiquitination of the TβRI intracellular domain is a prerequisite for TGFβ regulation of mRNA for cyclin D1 (CCND1), expression, as well as for the regulation of other genes controlling the cell cycle, differentiation, and invasiveness of prostate cancer cells.     

TGFβ in T cell biology and tumor immunity: Angel or devil?

The evolutionally conserved transforming growth factor β (TGFβ) affects multiple cell types in the immune system by either stimulating or inhibiting their differentiation and function. Studies using transgenic mice with ablation of TGFβ or its receptor have revealed the biological significance of TGFβ signaling in the control of T cells. However, it is now clear that TGFβ is more than an immunosuppressive cytokine. Disruption of TGFβ signaling pathway also leads to impaired generation of certain T cell populations. Therefore, in the normal physiological state, TGFβ actively maintains T cell homeostasis and regulates T cell function. However, in the tumor microenvironment, TGFβ creates an immunosuppressive milieu that inhibits antitumor immunity. Here, we review recent advances in our understanding of the roles of TGFβ in the regulation of T cells and tumor immunity.     

Periostin accelerates human malignant melanoma progression by modifying the melanoma microenvironment

Given no reliable therapy for advanced malignant melanoma, it is important to elucidate the molecular mechanisms underlying the disease progression. Using a quantitative proteomics approach, the ‘isobaric tags for relative and absolute quantitation (iTRAQ)’ method, we identified that the extracellular matrix protein, periostin (POSTN), was highly expressed in invasive melanoma compared with normal skin. An immunohistochemical analysis showed that POSTN was expressed in all invasive melanoma (n = 20) and metastatic lymph node (n = 5) tissue samples, notably restricted in their stroma. In terms of the intercellular regulation of POSTN, we found that there was upregulation of POSTN when melanoma cells and normal human dermal fibroblasts (NHDFs) were cocultured, with restricted expression of TGF‐β1 and TGF‐β3. In a functional analyses, recombinant and NHDF‐derived POSTN significantly accelerated melanoma cell proliferation via the integrin/mitogen‐activated protein kinase (MAPK) signaling pathway in vitro. The size of implanted melanoma tumors was significantly suppressed in POSTN/Rag2 double knockout mice compared with Rag2 knock‐out mice. These results indicate that NHDF‐derived POSTN accelerates melanoma progression and might be a promising therapeutic target for malignant melanoma.     

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.     

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.