Exploring Hypotheses of the Actions of TGF-β1 in Epidermal Wound Healing Using a 3D Computational Multiscale Model of the Human Epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-β1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-β1 literature–derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units (keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-β1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged (by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-β1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-β1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing.     

IKKalpha, a critical regulator of epidermal differentiation and a suppressor of skin cancer

IκB kinase α (IKKα), one of the two catalytic subunits of the IKK complex involved in nuclear factor κB (NF-κB) activation, also functions as a molecular switch that controls epidermal differentiation. This unexpected function requires IKKα nuclear translocation but does not depend on its kinase activity, and is independent of NF-κB signalling. Ikkα^–/– mice present with a hyperproliferative and undifferentiated epidermis characterized by complete absence of a granular layer and stratum corneum. Ikkα-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate even when subjected to differentiation-inducing stimuli. This antiproliferative function of IKKα is also important for the suppression of squamous cell carcinogenesis. The exact mechanisms by which nuclear IKKα controls keratinocyte proliferation and differentiation remained mysterious for some time. Recent studies, however, have revealed that IKKα is a major cofactor in a TGFβ–Smad2/3 signalling pathway that is Smad4 independent. This pathway controls cell cycle withdrawal during keratinocyte terminal differentiation. Although these are not the only functions of nuclear IKKα, this multifunctional protein is a key regulator of keratinocyte and epidermal differentiation and a critical suppressor of skin cancer.     

The Peritoneum Is Both a Source and Target of TGF-β in Women with Endometriosis

Transforming growth factor-β (TGF-β) is believed to play a major role in the aetiology of peritoneal endometriosis. We aimed to determine if the peritoneum is a source of TGF-β and if peritoneal TGF-β expression, reception or target genes are altered in women with endometriosis. Peritoneal fluid, peritoneal bushings and peritoneal biopsies were collected from women with and without endometriosis. TGF-β1, 2 and 3 protein concentrations were measured in the peritoneal fluid. TGF-β1 was measured in mesothelial cell conditioned media. Control peritoneum and peritoneum prone to endometriosis (within Pouch of Douglas) from women without disease (n = 16) and peritoneum distal and adjacent to endometriosis lesions in women with endometriosis (n = 15) and were analysed for TGF-β expression, reception and signalling by immunohistochemistry, qRT-PCR and a TGF-β signalling PCR array. TGF-β1 was increased in the peritoneal fluid of women with endometriosis compared to those without disease (P<0.05) and peritoneal mesothelial cells secrete TGF-β1 in-vitro. In women with endometriosis, peritoneum from sites adjacent to endometriosis lesions expressed higher levels of TGFB1 mRNA when compared to distal sites (P<0.05). The TGF-β-stimulated Smad 2/3 signalling pathway was active in the peritoneum and there were significant increases (P<0.05) in expression of genes associated with tumorigenesis (MAPK8, CDC6), epithelial-mesenchymal transition (NOTCH1), angiogenesis (ID1, ID3) and neurogenesis (CREB1) in the peritoneum of women with endometriosis. In conclusion, the peritoneum, and in particular, the peritoneal mesothelium, is a source of TGF-β1 and this is enhanced around endometriosis lesions. The expression of TGF-β-regulated genes is altered in the peritoneum of women with endometriosis and this may promote an environment favorable to lesion formation.     

Endoglin mediates fibronectin/α5β1 integrin and TGF-β pathway crosstalk in endothelial cells

Both the transforming growth factor β (TGF-β) and integrin signalling pathways have well-established roles in angiogenesis. However, how these pathways integrate to regulate angiogenesis is unknown. Here, we show that the extracellular matrix component, fibronectin, and its cellular receptor, α5β1 integrin, specifically increase TGF-β1- and BMP-9-induced Smad1/5/8 phosphorylation via the TGF-β superfamily receptors endoglin and activin-like kinase-1 (ALK1). Fibronectin and α5β1 integrin increase Smad1/5/8 signalling by promoting endoglin/ALK1 cell surface complex formation. In a reciprocal manner, TGF-β1 activates α5β1 integrin and downstream signalling to focal adhesion kinase (FAK) in an endoglin-dependent manner. α5β1 integrin and endoglin form a complex on the cell surface and co-internalize, with their internalization regulating α5β1 integrin activation and signalling. Functionally, endoglin-mediated fibronectin/α5β1 integrin and TGF-β pathway crosstalk alter the responses of endothelial cells to TGF-β1, switching TGF-β1 from a promoter to a suppressor of migration, inhibiting TGF-β1-mediated apoptosis to promote capillary stability, and partially mediating developmental angiogenesis in vivo. These studies provide a novel mechanism for the regulation of TGF-β superfamily signalling and endothelial function through crosstalk with integrin signalling pathways.     

Transforming Growth Factor-β3 (TGF-β3) Knock-in Ameliorates Inflammation Due to TGF-β1 Deficiency While Promoting Glucose Tolerance

Three homologues of TGF-β exist in mammals as follows: TGF-β1, TGF-β2, and TGF-β3. All three proteins share high homology in their amino acid sequence, yet each TGF-β isoform has unique heterologous motifs that are highly conserved during evolution. Although these TGF-β proteins share similar properties in vitro, isoform-specific properties have been suggested through in vivo studies and by the unique phenotypes for each TGF-β knock-out mouse. To test our hypothesis that each of these homologues has nonredundant functions, and to identify such isoform-specific roles, we genetically exchanged the coding sequence of the mature TGF-β1 ligand with a sequence from TGF-β3 using targeted recombination to create chimeric TGF-β1/3 knock-in mice (TGF-β1^Lβ3/Lβ3). In the TGF-β1^Lβ3/Lβ3 mouse, localization and activation still occur through the TGF-β1 latent associated peptide, but cell signaling is triggered through the TGF-β3 ligand that binds to TGF-β receptors. Unlike TGF-β1^−/− mice, the TGF-β1^Lβ3/Lβ3 mice show neither embryonic lethality nor signs of multifocal inflammation, demonstrating that knock-in of the TGF-β3 ligand can prevent the vasculogenesis defects and autoimmunity associated with TGF-β1 deficiency. However, the TGF-β1^Lβ3/Lβ3 mice have a shortened life span and display tooth and bone defects, indicating that the TGF-β homologues are not completely interchangeable. Remarkably, the TGF-β1^Lβ3/Lβ3 mice display an improved metabolic phenotype with reduced body weight gain and enhanced glucose tolerance by induction of beneficial changes to the white adipose tissue compartment. These findings reveal both redundant and unique nonoverlapping functional diversity in TGF-β isoform signaling that has relevance to the design of therapeutics aimed at targeting the TGF-β pathway in human disease.     

miR-58 family and TGF-β pathways regulate each other in Caenorhabditis elegans

Despite the fact that microRNAs (miRNAs) modulate the expression of around 60% of protein-coding genes, it is often hard to elucidate their precise role and target genes. Studying miRNA families as opposed to single miRNAs alone increases our chances of observing not only mutant phenotypes but also changes in the expression of target genes. Here we ask whether the TGF-β signalling pathways, which control many animal processes, might be modulated by miRNAs in Caenorhabditis elegans. Using a mutant for four members of the mir-58 family, we show that both TGF-β Sma/Mab (controlling body size) and TGF-β Dauer (regulating dauer, a stress-resistant larval stage) are upregulated. Thus, mir-58 family directly inhibits the expression of dbl-1 (ligand), daf-1, daf-4 and sma-6 (receptors) of TGF-β pathways. Epistasis experiments reveal that whereas the small body phenotype of the mir-58 family mutant must invoke unknown targets independent from TGF-β Sma/Mab, its dauer defectiveness can be rescued by DAF-1 depletion. Additionally, we found a negative feedback loop between TGF-β Sma/Mab and mir-58 and the related mir-80. Our results suggest that the interaction between mir-58 family and TGF-β genes is key on decisions about animal growth and stress resistance in C. elegans and perhaps other organisms.     

TGF-β Isoform Specific Regulation of Airway Inflammation and Remodelling in a Murine Model of Asthma

The TGF-β family of mediators are thought to play important roles in the regulation of inflammation and airway remodelling in asthma. All three mammalian isoforms of TGF-β, TGF-β_1–3, are expressed in the airways and TGF-β₁ and -β₂ are increased in asthma. However, there is little information on the specific roles of individual TGF-β isoforms. In this study we assess the roles of TGF-β₁ and TGF-β₂ in the regulation of allergen-induced airway inflammation and remodelling associated with asthma, using a validated murine model of ovalbumin sensitization and challenge, and isoform specific TGF-β neutralising antibodies. Antibodies to both isoforms inhibited TGF-β mediated Smad signalling. Anti-TGF-β₁ and anti-TGF-β₂ inhibited ovalbumin-induced sub-epithelial collagen deposition but anti-TGF-β₁ also specifically regulated airway and fibroblast decorin deposition by TGF-β₁. Neither antibody affected the allergen-induced increase in sub-epithelial fibroblast-like cells. Anti- TGF-β₁ also specifically inhibited ovalbumin-induced increases in monocyte/macrophage recruitment. Whereas, both TGF-β₁ and TGF-β₂ were involved in regulating allergen-induced increases in eosinophil and lymphocyte numbers. These data show that TGF-β₁ and TGF-β₂ exhibit a combination of specific and shared roles in the regulation of allergen-induced airway inflammation and remodelling. They also provide evidence in support of the potential for therapeutic regulation of specific subsets of cells and extracellular matrix proteins associated with inflammation and remodelling in airway diseases such as asthma and COPD, as well as other fibroproliferative diseases.     

Inhibition of Contractile Function in Human Joint Capsule Myofibroblasts by Targeting the TGF-β1 and PDGF Pathways

Background

Contractile myofibroblasts (MFs) accumulate in the joint capsules of patients suffering from posttraumatic joint stiffness. MF activation is controlled by a complex local network of growth factors and cytokines, ending in the increased production of extracellular matrix components followed by soft tissue contracture. Despite the tremendous growth of knowledge in this field, inconsistencies remain in practice and prevention.

Methods and Findings

In this in vitro study, we isolated and cultured alpha-smooth muscle actin (α-SMA) positive human joint capsule MFs from biopsy specimens and investigated the effect of profibrotic and antifibrotic agents on MF function. Both TGF-β1 and PDGF significantly induced proliferation and increased extracellular matrix contraction in an established 3D collagen gel contraction model. Furthermore, both growth factors induced α-SMA and collagen type I gene expression in MFs. TGF-β1 down-regulated TGF-β1 and TGF-β receptor (R) 1 and receptor (R) 2 gene expression, while PDGF selectively down-regulated TGF-β receptor 2 gene expression. These effects were blocked by suramin. Interestingly, the anti-oxidant agent superoxide dismutase (SOD) blocked TGF-β1 induced proliferation and collagen gel contraction without modulating the gene expression of α-SMA, collagen type I, TGF-β1, TGF-β R1 and TGF-β R2.

Conclusions

Our results provide evidence that targeting the TGF-β1 and PDGF pathways in human joint capsule MFs affects their contractile function. TGF-β1 may modulate MF function in the joint capsule not only via the receptor signalling pathway but also by regulating the production of profibrotic reactive oxygen species (ROS). In particular, anti-oxidant agents could offer promising options in developing strategies for the prevention and treatment of posttraumatic joint stiffness in humans.     

Biomarkers of TGF-β Signaling Pathway and Prognosis of Pancreatic Cancer

Background

Transforming growth factor (TGF)-β signaling pathway, may act both as a tumor suppressor and as a tumor promoter in pancreatic cancer, depending on tumor stage and cellular context. TGF-β pathway has been under intensive investigation as a potential therapeutic target in the treatment of cancer. We hypothesized a correlation between TGF-βR2/SMAD4 expression in the tumor, plasma TGF-β1 ligand level, genetic variation in TGF-B pathway and prognosis of pancreatic cancer.

Method

We examined TGF-βR2 and SMAD4 protein expression in biopsy or surgical samples from 91 patients with pancreatic ductal adenocarcinoma (PDAC) using immunohistochemistry. Plasma level of TGF-β1 was measured in 644 patients with PDAC using ELISA. Twenty-eight single nucleotide polymorphisms (SNP) of the TGF-β1, TGF-β2, TGF-β3, TGF-βR1, TGF-βR2, and SMAD4 genes were determined in 1636 patients with PDAC using the Sequenom method. Correlation between protein expression in the tumor, plasma TGF-β1 level, and genotypes with overall survival (OS) was evaluated with Cox proportional regression models.

Results

The expression level of TGF-βR2 and SMAD4 as an independent marker was not associated with OS. However, patients with both low nuclear staining of TGF-βR2 and high nuclear staining of SMAD4 may have better survival (P = 0.06). The mean and median level of TGF-β1 was 15.44 (SD: 10.99) and 12.61 (interquartile range: 8.31 to 19.04) ng/ml respectively. Patients with advanced disease and in the upper quartile range of TGF-β1 level had significantly reduced survival than those with low levels (P = 0.02). A significant association of SMAD4 SNP rs113545983 with overall survival was observed (P<0.0001).

Conclusion

Our data provides valuable baseline information regarding the TGF-β pathway in pancreatic cancer, which can be utilized in targeted therapy clinical trials. High TGF-β1 plasma level, SMAD4 SNP or TGF-βR2/SMAD4 tumor protein expression may suggest a dependence on this pathway in patients with advanced pancreatic cancer.     

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.     

TGF-β Signalling Is Required for CD4+ T Cell Homeostasis But Dispensable for Regulatory T Cell Function

TGF-β is widely held to be critical for the maintenance and function of regulatory T (T_reg) cells and thus peripheral tolerance. This is highlighted by constitutive ablation of TGF-β receptor (TR) during thymic development in mice, which leads to a lethal autoimmune syndrome. Here we describe that TGF-β–driven peripheral tolerance is not regulated by TGF-β signalling on mature CD4⁺ T cells. Inducible TR2 ablation specifically on CD4⁺ T cells did not result in a lethal autoinflammation. Transfer of these TR2-deficient CD4⁺ T cells to lymphopenic recipients resulted in colitis, but not overt autoimmunity. In contrast, thymic ablation of TR2 in combination with lymphopenia led to lethal multi-organ inflammation. Interestingly, deletion of TR2 on mature CD4⁺ T cells does not result in the collapse of the T_reg cell population as observed in constitutive models. Instead, a pronounced enlargement of both regulatory and effector memory T cell pools was observed. This expansion is cell-intrinsic and seems to be caused by increased T cell receptor sensitivity independently of common gamma chain-dependent cytokine signals. The expression of Foxp3 and other regulatory T cells markers was not dependent on TGF-β signalling and the TR2–deficient T_reg cells retained their suppressive function both in vitro and in vivo. In summary, absence of TGF-β signalling on mature CD4⁺ T cells is not responsible for breakdown of peripheral tolerance, but rather controls homeostasis of mature T cells in adult mice.     

Oligodendrocyte Precursor Cells Support Blood-Brain Barrier Integrity via TGF-β Signaling

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-β1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-β-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-β1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-β1 deficient mice (OPC-specific TGF-β1 partial KO mice: Pdgfra^Cre/Tgfb1^flox/wt mice or OPC-specific TGF-β1 total KO mice: Pdgfra^Cre/Tgfb1^flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-β signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.     

Osteopontin Is Induced by TGF-β2 and Regulates Metabolic Cell Activity in Cultured Human Optic Nerve Head Astrocytes

The aqueous humor (AH) component transforming growth factor (TGF)-β2 is strongly correlated to primary open-angle glaucoma (POAG), and was shown to up-regulate glaucoma-associated extracellular matrix (ECM) components, members of the ECM degradation system and heat shock proteins (HSP) in primary ocular cells. Here we present osteopontin (OPN) as a new TGF-β2 responsive factor in cultured human optic nerve head (ONH) astrocytes. Activation was initially demonstrated by Oligo GEArray microarray and confirmed by semiquantitative (sq) RT-PCR, realtime RT-PCR and western blot. Expressions of most prevalent OPN receptors CD44 and integrin receptor subunits αV, α4, α 5, α6, α9, β1, β3 and β5 by ONH astrocytes were shown by sqRT-PCR and immunofluorescence labeling. TGF-β2 treatment did not affect their expression levels. OPN did not regulate gene expression of described TGF-β2 targets shown by sqRT-PCR. In MTS-assays, OPN had a time- and dose-dependent stimulating effect on the metabolic activity of ONH astrocytes, whereas TGF-β2 significantly reduced metabolism. OPN signaling via CD44 mediated a repressive outcome on metabolic activity, whereas signaling via integrin receptors resulted in a pro-metabolic effect. In summary, our findings characterize OPN as a TGF-β2 responsive factor that is not involved in TGF-β2 mediated ECM and HSP modulation, but affects the metabolic activity of astrocytes. A potential involvement in a protective response to TGF-β2 triggered damage is indicated, but requires further investigation.     

Continuous Cyclic Mechanical Tension Increases Ank Expression in Endplate Chondrocytes Through the TGF-β1 and p38 Pathway

The normal ANK protein has a strong influence on anti-calcification. It is known that TGF-β1 is also able to induce extracellular inorganic pyrophosphate (ePPi) elaboration via the TGF-β1-induced ank gene expression and the mitogen-activated protein kinase (MAPK) signaling acts as a downstream effector of TGF-β1. We hypothesized that the expression of the ank gene is regulated by mechanics through TGF-β1-p38 pathway. In this study, we investigated the mechanism of short-time mechanical tension-induced ank gene expression. We found that the continuous cyclic mechanical tension (CCMT) increased the ank gene expression in the endplate chondrocytes, and there was an increase in the TGF-β1 expression after CCMT stimulation. The ank gene expression significantly increased when treated by TGF-β1 in a dose-dependent manner and decreased when treated by SB431542 (ALK inhibitor) in a dose-dependent manner. Our study results indicate that CCMT-induced ank gene expressions may be regulated by TGF-β1 and p38 MAPK pathway.Key words: Continuous cyclic mechanical tension (CCMT), Endplate chondrocytes, ank, TGF-β1, p38     

Association between Fibrillin1 Polymorphisms (rs2118181, rs10519177) and Transforming Growth Factor β1 Concentration in Human Plasma

Transforming growth factor (TGF)-β1 is a cytokine that participates in a broad range of cellular regulatory processes and is associated with various diseases including aortic aneurysm. Increased TGF-β1 levels are linked to Marfan syndrome (MFS) caused by fibrillin1 (FBN1) mutations and subsequent defects in signaling system. FBN1 single nucleotide polymorphisms (SNPs) rs2118181 and rs1059177 do not cause MFS but are associated with dilative pathology of aortic aneurysms (DPAAs). TGF-β1 and FBN1 SNPs rs2118181 and rs1059177 are potential biomarkers for early diagnosis of DPAA. We investigated the relationship between TGF-β1 levels in human blood plasma and FBN1 rs2118181 and rs1059177 in 269 individuals. The results showed a quantitative dependence of SNP genotype and TGF-β1 concentration. Presence of a single rs2118181 minor allele (G) increased the amount of TGF-β1 by roughly 1 ng/mL. Two copies of FBN1 rs1059177 minor allele (G) were required to have an additive effect on TGF-β1 levels. We found higher TGF-β1 concentrations in men compared with women (p = 0.001). A strong correlation between TGF-β1 levels and FBN1 SNPs suggests that a single nucleotide substitution in FBN1 sequence might reduce bioavailability or binding properties of fibrillin-1 and have an effect on TGF-β1 activation and cytokine concentration in blood plasma. By establishing the relationship between TGF-β1 and FBN1 SNPs rs2118181 and rs1059177, we provide evidence that their combination might be used as molecular biomarkers to identify patients at risk for sporadic ascending aortic aneurysm and aortic dissection.     

High glucose–induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model

Background: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. Methods: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. Results: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition. Conclusions: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.     

Notch and TGF-β pathways cooperatively regulate receptor protein tyrosine phosphatase-κ (PTPRK) gene expression in human primary keratinocytes

Receptor protein tyrosine phosphatase-κ (PTPRK) specifically and directly dephosphorylates epidermal growth factor receptor (EGFR), thereby limiting EGFR function in primary human keratinocytes. PTPRK expression is increased by the TGF-β/Smad3 pathway and cell–cell contact. Because the Notch receptor pathway is responsive to cell–cell contact and regulates keratinocyte growth and differentiation, we investigated the interplay between Notch and TGF-β pathways in regulation of PTPRK expression in human keratinocytes. Suppression of Notch signaling by γ-secretase inhibitors substantially reduced cell contact induction of PTPRK gene expression. In sparse keratinocyte cultures, addition of soluble Notch-activating ligand jagged one peptide (Jag1) induced PTPRK. Of interest, cell contact–induced expression of TGF-β1 and TGF-β receptor inhibitor SB431542 inhibited contact-induced expression of PTPRK. Furthermore, inhibition of Notch signaling, via knockdown of Notch1 or by γ-secretase inhibitors, significantly reduced TGF-β–induced PTPRK gene expression, indicating that Notch and TGF-β pathways function together to regulate PTPRK. Of importance, the combination of Jag1 plus TGF-β results in greater PTPRK expression and lower EGFR tyrosine phosphorylation than either ligand alone. These data indicate that Notch and TGF-β act in concert to stimulate induction of PTPRK, which suppresses EGFR activation in human keratinocytes.     

The Four-Herb Chinese Medicine ANBP Enhances Wound Healing and Inhibits Scar Formation via Bidirectional Regulation of Transformation Growth Factor Pathway

The four-herb Chinese medicine ANBP is a pulverized mixture of four herbs including Agrimonia Eupatoria (A), Nelumbo Nucifera Gaertn (N), Boswellia Carteri (B) and Pollen Typhae Angustifoliae (P). The combination of the four herbs was first described in Chinese canonical medicine about 2000 years ago for treatment of various trauma disorders, such as hemostasis, antiinflammatory, analgesia, and wound healing, etc. However, the precise mechanisms of ANBP are still unclear. In our study, using rabbit ear hypertrophic scar models of full-thickness skin defect, we showed that local ANBP treatment not only significantly enhanced wound healing by relieving inflammation, increasing formation of granulation tissue and accelerating re-epithelialization, but also reduced scar formation by decreasing collagen production, protuberant height and volume of scars, and increasing collagen maturity. We demonstrated that these effects of ANBP are associated with transforming growth factor (TGF)-β1-mediated signalling pathways through Smad-dependent pathways. ANBP treatment significantly increased expression of TGF-β1 and Smad2/3 mRNA at the early stage of wound healing, and led to markedly decrease expression of TGF-β1 and Smad2/3 compared with the control group after 14 days post-wounding. Taken together, our results defined a bidirectional regulation role of ANBP for TGF-β1/Smad pathway in promoting wound healing and alleviating scar formation, which may be an effective therapy for human wounds at the earliest stage.