Smad-dependent Signaling Research Articles

Graphene Quantum Dots as Antifibrotic Therapy for Kidney Disease.

Graphene quantum dots (GQDs) have received much attention for their biomedical applications, such as bioimaging and drug delivery. Additionally, they have antioxidant and anti-inflammatory properties. We used GQDs to treat renal fibrosis and confirmed their ability to protect renal cells from excessive oxidative stress in vitro and in vivo. Tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in 7- to 8-week-old male C57BL/6 mice. GQDs were administered by intravenous injection to mimic clinical treatment. The levels of oxidative stress, ROS production, apoptosis and proinflammatory cytokines and the activity of the TGFβ1/Smad pathway were evaluated after treatment with GQDs. In vitro, rhTGF-β1 was used to induce fibrosis in primary kidney tubule epithelial cells. GQDs alleviated fibrosis and morphological changes after UUO induction. At the mRNA and protein levels, GQDs significantly reduced the expression of fibrotic markers and proinflammatory cytokines, decreased ROS production and TGF-β1 expression, and affected Smad-dependent signaling pathways. In vitro, GQDs inhibited rhTGF-β1-induced epithelial-to-mesenchymal transition in primary kidney tubule epithelial cells and reduced apoptosis and ROS accumulation. This study revealed the role of GQDs in kidney fibrosis: GQDs effectively attenuated major fibrogenesis events by inhibiting ROS accumulation and the vicious cycle of the ROS and TGF-β1/Smad signaling pathways, as well as alleviating cell apoptosis and inflammation. Thus, GQDs may be a therapeutic option for chronic kidney disease progression.

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

8786 Novel TGFbeta Ligand Regulated Genes in WT and FSTL3 KO Testis Identified by ChIP-Seq Analysis: Implications for Signalling Cross-talk

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Folistatin Like 3 (FSTL3) is a known antagonist of activin and myostatin action that when deleted from the mouse genome displayed a set of phenotypic changes that are directly linked to glucose metabolism and reproductive function. Although our previous work showed that β-cell hyperplasia, increased pancreatic islet number and size and reduced visceral fat seen in FSTL3 knockout mice (FSTL3 KO) could be explained by enhanced activin and myostatin action, the molecular bases of the changes in the reproductive function seen in this mouse model remain elusive. In contrast to testicular degeneration produced by INHBA (activin A) transgenic overexpression in mice, deletion of FSTL3 results in increased testicular size, with increased Sertoli cell numbers and early meiotic entry of the first spermatogenic wave. This suggests a possibility that the phenotype in FSTL3 deleted mice might involve alteration of additional transcripts other than those induced by activin alone in the testis. To identify transcripts induced in FSTL3 deleted testis as a consequence of induced TGFβ ligand action we performed sequencing of Smad4 Chromatin immunoprecipitation (ChIP-Seq) from postnatal (3d) and post-pubertal (56d) whole testis. Our results show high activity of Smad4 signalling during postnatal stages compared to post-pubertal stages in both genotypes (Fstl3-KO vs wild-type). Interestingly, postnatal testis from FSTL3 KO contains significantly less activity from Smad4 signalling (69 peaks) compared to wild type (775 peaks). By contrast, on the whole, Smad4 signalling in post-pubertal stages is less active with 34 peaks in wild-type and 29 peaks in Fstl3-KO of which 8 genes are SMAD activated in both genotypes. To identify whether SMAD-dependent signalling produces significant differences in RNA expression, we then used the list of genes that generated peaks in the Smad4-ChIP-Seq experiment to filter our RNA-Seq data generated in a parallel experiment followed by differential gene expression analysis of GO terms by GSEA in postnatal testis from both genotypes. We found that Smad4-interacting genes in posnatal FSTL3 KO testis generate enrichment of 13 pathways including the androgen receptor signalling pathway. Taken together so far, our findings reveal a novel catalogue of SMAD-regulated genes in the testis, identifies a set of genes specifically upregulated in the testis in a SMAD-dependent manner upon FSTL3 deletion and also identify possible cross-talk between TGFβ and 13 other signalling pathways. Currently we are further analysing our data to identify whether there are SMAD regulated genes and pathways common in postnatal and postpubertal testes. We are also undertaking experiments to investigate expression pattern in FSTL3 KO testis to identify which pathways might specifically affect Sertoli cell proliferation and activity. Presentation: 6/2/2024

Discovery of Two Highly Selective Structurally Orthogonal Chemical Probes for Activin Receptor-like Kinases 1 and 2.

Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.

Dissecting TGF-β-induced glioblastoma invasion with engineered hyaluronic acid hydrogels.

Glioma stem cells (GSCs) contribute to rapid cellular invasion in glioblastoma (GBM). Transforming growth factor-β (TGF-β) has been strongly implicated in supporting key GSC functions, including stemness, immunosuppression, and resistance. Although TGF-β is well-known as a driver of cancer invasion, how TGF-β supports the invasion of GSCs is not well understood. Progress in understanding mechanisms of TGF-β-driven invasion in GSC-derived tumors has been limited by an absence of three-dimensional (3D) culture systems that support TGF-β-stimulated invasion. Here, we show that 3D hyaluronic acid (HA) matrices can address this need. We perform bioinformatic analysis of human glioma datasets, which reveals progressive enrichment of TGF-β-related gene expression with increasingly aggressive glioma grade and GBM subtype. We then experimentally screen the invasion of a panel of human GSC spheroids through a set of 3D matrix systems, including collagen I, Matrigel, and HA, and find that only HA recapitulates TGF-β-induced invasion. We then show that GSCs differ in their ability to invade HA in a way that can be predicted from TGF-β receptor 2 expression and SMAD2 phosphorylation. GSC spheroid invasion depends strongly on the presence of RGD peptides on the HA backbone but is surprisingly independent of matrix metalloprotease degradability. Finally, we demonstrate that TGF-β stimulates invasion through SMAD-dependent signaling, consistent with recent observations that TGF-β/SMAD signals drive tumor microtube formation and invasion. Our work supports further development of HA as a matrix platform for dissecting contributions of TGF-β and other cytokines to GBM invasion and screening of cytokine-dependent invasion in human tumors.

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

YTHDF2 governs muscle size through a targeted modulation of proteostasis

The regulation of proteostasis is fundamental for maintenance of muscle mass and function. Activation of the TGF-β pathway drives wasting and premature aging by favoring the proteasomal degradation of structural muscle proteins. Yet, how this critical post-translational mechanism is kept in check to preserve muscle health remains unclear. Here, we reveal the molecular link between the post-transcriptional regulation of m6A-modified mRNA and the modulation of SMAD-dependent TGF-β signaling. We show that the m6A-binding protein YTHDF2 is essential to determining postnatal muscle size. Indeed, muscle-specific genetic deletion of YTHDF2 impairs skeletal muscle growth and abrogates the response to hypertrophic stimuli. We report that YTHDF2 controls the mRNA stability of the ubiquitin ligase ASB2 with consequences on anti-growth gene program activation through SMAD3. Our study identifies a post-transcriptional to post-translational mechanism for the coordination of gene expression in muscle.

Mesenchymal stem cells derived from CHIR99021 and TGF‑β induction remained on the colicomentum and improved cardiac function of a rat model of acute myocardium infarction.

Human induced pluripotent stem cells (hiPSCs) have been regarded as a potential stem cell source for cell therapy. However, the production of cells with mesenchymal potential from hiPSCs through spontaneous differentiation is time consuming and laborious. In the present study, the combined use of the GSK-3 inhibitor CHIR99021 and TGF-β was used to obtain mesenchymal stem cell (MSC)-like cells from hiPSCs. During the induction process, the transcription of epithelial-mesenchymal transition (EMT)-related genes N-cadherin and Vimentin in the transformed cells was upregulated, whereas the transcription of E-cadherin and pluripotency-related transcription factors SOX2, OCT4 and NANOG did not change significantly. This indicated that whilst cells were pluripotent, EMT was initiated by the upregulation of transcription of EMT promoting genes. Both SMAD-dependent and independent signalling pathways were significantly activated by the combined induction treatment compared with the single factor induction. The hiPSC-derived MSC-like cells (hiPSC-MSCs) expressed MSC-related markers and acquired osteogenic, chondrogenic and adipogenic differentiation potentials. After being injected into the peritoneal cavity of rats, the hiPSC-MSCs secreted angiogenic and immune-regulatory factors and remained on the colicomentum for 3 weeks. Within an 11-week period, four intraperitoneal hiPSC-MSC injections (1x107 cells/injection) into acute myocardial infarction (AMI) model rats significantly increased the left ventricular ejection fraction, left ventricular fractional shortening and angiogenesis and significantly reduced scar size and the extent of apoptosis in the infarcted area compared with that of the control PBS injection. Symptoms of hiPSC-MSC-induced immune reaction or tumour formation were not observed over the course of the experiment in the hiSPC-MSC treated rats. In conclusion, the CHIR99021 and TGF-β combined induction was a rapid and effective method to obtain MSC-like cells from hiPSCs and multiple high dose intraperitoneal injections of hiPSC-derived MSCs were safe and effective at restoring cardiac function in an AMI rat model.

Targeting SMAD-Dependent Signaling: Considerations in Epithelial and Mesenchymal Solid Tumors.

SMADs are the canonical intracellular effector proteins of the TGF-β (transforming growth factor-β). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-β/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-β/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors.

Upregulated LncRNA-Meg3 modulates the proliferation and survival of MEPM cells via interacting with Smad signaling in TCDD-induced cleft palate

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in utero can result in high rates of cleft palate (CP) formation, yet the underlying mechanisms remain to be characterized. In vivo, the lncRNA Meg3 was upregulated following TCDD treatment in CP-associated murine embryonic palatal tissue, with concomitant changes in proliferative and apoptotic activity in these murine embryonic palatal mesenchymal (MEPM) cells. Meg3 can modulate the TGF-β/Smad to control the proliferation, survival, and differentiation of cells. Accordingly, TCCD and TGF-β1 were herein used to treat MEPM cells in vitro, revealing that while TCDD exposure altered the proliferative activity and apoptotic death of these cells, exogenous TGF-β1 exposure antagonized these effects via TGF-β/Smad signaling. TCDD promoted Meg3 upregulation, whereas TGF-β1 suppressed TCDD-driven upregulation of this lncRNA. Meg3 was additionally determined to directly interact with Smad2, with significant Meg3 enrichment in Smad2-immunoprecipitates following TCDD treatment. When Meg3 was silenced, the impact of TCDD on Smad signaling, proliferative activity, and apoptosis were ablated, while the effects of exogenous TGF-β1 were unchanged. This supports a model wherein Meg3 is upregulated in TCDD-exposed palatal tissue whereupon it can interact with Smad2 to suppress Smad-dependent signaling, thus controlling MEPM cell proliferation and apoptosis, contributing to TCDD-induced CP, which provides a theoretical support for the precautions of cleft palate induced by TCDD.

The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFβ signaling and cancer cell invasion

BackgroundLong non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor β (TGFβ) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFβ targets, yet, their mechanism of action and biological role in cancer remain poorly understood.MethodsWhole-genome transcriptomics identified lncRNA genes regulated by TGFβ. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFβ-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue.ResultsTranscriptomics of TGFβ signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFβ led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFβ, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFβ stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues.ConclusionsLINC00707 interacts with Smad proteins and limits the output of TGFβ signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion.EZy4eavcmbb38C5oFYKmkjVideo

Impact of the transforming growth factor-β pathway on vascular restenosis and its mechanism.

As a crucial regulatory molecule in the context of vascular stenosis, transforming growth factor-β (TGF-β), plays a pivotal role in its initiation and progression. TGF-β, a member of the TGF-β superfamily, can bind to the TGF-β receptor and transduce extracellular to intracellular signals through canonical Smad dependent or noncanonical signaling pathways to regulate cell growth, proliferation, differentiation, and apoptosis. Restenosis remains one of the most challenging problems in cardiac, cerebral, and peripheral vascular disease worldwide. The mechanisms for occurrence and development of restenosis are diverse and complex. The TGF-β pathway exhibits diversity across various cell types. Hence, clarifying the specific roles of TGF-β within different cell types and its precise impact on vascular stenosis provides strategies for future research in the field of stenosis.

Enhanced BMP signaling leads to enlarged nasal cartilage formation in mice

Bone morphogenetic proteins (BMPs) are required for craniofacial bone development. However, it remains elusive how BMP signaling regulates craniofacial cartilage development. To address this question, we utilized a genetic system to enhance BMP signaling via one of BMP type I receptors ALK2 in a chondrocyte-specific manner (hereafter Ca-Alk2:Col2-Cre) in mice. Ca-Alk2:Col2-Cre mice died shortly after birth due to severe craniofacial abnormalities including cleft palate, defective tongue, and shorter mandible formation. Histological analysis revealed that these phenotypes were attributed to the extensive chondrogenesis. Compared with controls, enhanced SOX9 and RUNX2 production were observed in nasal cartilage of Ca-Alk2:Col2-Cre mice. To reveal the mechanisms responsible for enlarged nasal cartilage, we examined Smad-dependent and Smad-independent BMP signaling pathways. While the Smad-independent BMP signaling pathway including p38, ERK, and JNK remained silent, the Smad1/5/9 was highly phosphorylated in Ca-Alk2:Col2-Cre mice. Interestingly, Ca-Alk2:Col2-Cre mice showed enhanced S6 kinase phosphorylation, a readout of mammalian target of rapamycin complex 1 (mTORC1). These findings may suggest that enhanced Smad-dependent BMP signaling positively regulates the mTOR pathway and stimulates chondrocytes toward hypertrophic differentiation, thereby leading to enlarged nasal cartilage formation in mice.

Abstract P2051: Fibroblast-specific TGF-b Signaling Promotes Interstitial Fibrosis, Cardiomyocyte Hypertrophy And Cardiac Dysfunction In The Diabetic Heart Through Smad3 Activation

Objectives: Patients with diabetes exhibit a high prevalence of HFpEF associated with interstitial fibrosis and cardiomyocyte hypertrophy. However, the cellular and molecular mechanisms involved in diabetic heart disease remain poorly understood. TGF-bs are potent fibrogenic mediators that are activated in diabetic hearts, and act through a single type 2 receptor, TbRII. We hypothesized that fibroblast-specific actions of TGF-bs may mediate fibrosis and dysfunction in diabetic hearts. Methods and Results: To test the hypothesis, we developed lean and db/db type 2 diabetic TbRII KO mice (FTbRII2KO) using the Col1a2 CreER driver. Fibroblast-specific loss of TGF-b signaling attenuated hypertrophy in db/db mouse hearts. Speckle-tracking echocardiography with strain imaging showed that fibroblast-specific TbRII loss improved systolic and diastolic cardiac function in diabetic animals. In contrast, fibroblast TbRII disruption caused a worsening of both cardiac systolic and diastolic function in lean animals. Attenuated dysfunction in diabetic mice lacking fibroblast TGF-b signaling was associated with reduced collagen fiber thickness and with decreased cardiomyocyte size, suggesting paracrine effects of fibroblasts on cardiomyocytes. To study the downstream cascade responsible for the effects of TGF-b, we generated lean and diabetic mice with fibroblast-specific Smad3 loss. In db/db mice, fibroblast-specific Smad3 loss phenocopied the effects of fibroblast-specific TbRII disruption, suggesting that in diabetic cardiac fibroblasts TGF-b acts through Smad3 signaling. The bioinformatic analyses of transcriptomic profiles of cardiac fibroblasts identified thrombospondin 4 as a candidate mediator of Smad3-dependent diabetes-associated activation. Conclusions: Fibroblast-specific TGF-b actions in diabetes promote cardiac dysfunction through effects on collagen deposition and via paracrine actions on cardiomyocytes. The effects of TGF-b on diabetic cardiac fibroblasts are Smad3-dependent. In contrast to the deleterious effects of fibroblast TGFb/Smad3 signaling in diabetic hearts, TGF-b-activated Smad3-dependent signaling is important to maintain homeostatic function and geometry in normal lean animals.

Connective tissue growth factor: Role in trabecular meshwork remodeling and intraocular pressure lowering.

Connective tissue growth factor (CTGF) is a distinct signaling molecule modulating many physiological and pathophysiological processes. This protein is upregulated in numerous fibrotic diseases that involve extracellular matrix (ECM) remodeling. It mediates the downstream effects of transforming growth factor beta (TGF-β) and is regulated via TGF-β SMAD-dependent and SMAD-independent signaling routes. Targeting CTGF instead of its upstream regulator TGF-β avoids the consequences of interfering with the pleotropic effects of TGF-β. Both CTGF and its upstream mediator, TGF-β, have been linked with the pathophysiology of glaucomatous optic neuropathy due to their involvement in the regulation of ECM homeostasis. The excessive expression of these growth factors is associated with glaucoma pathogenesis via elevation of the intraocular pressure (IOP), the most important risk factor for glaucoma. The raised in the IOP is due to dysregulation of ECM turnover resulting in excessive ECM deposition at the site of aqueous humor outflow. It is therefore believed that CTGF could be a potential therapeutic target in glaucoma therapy. This review highlights the CTGF biology and structure, its regulation and signaling, its association with the pathophysiology of glaucoma, and its potential role as a therapeutic target in glaucoma management.

Computational and Experimental Analyses for Pathogenicity Prediction of ACVRL1 Missense Variants in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease caused by the defects of ALK1/ACVRL1 receptor signaling. In this study, we evaluated 25 recently identified ACVRL1 missense variants using multiple computational pathogenicity classifiers and experimentally characterized their signal transduction capacity. Three extracellular residue variants showed no detectable cell surface expression and impairment of bone morphogenetic protein 9 (BMP9) responsiveness of SMAD-dependent transcription in luciferase assays. Four variants with amino acid replacement in the motifs essential for the intracellular kinase function lost SMAD-dependent signaling. Most of other variations in the kinase domain also caused marked downregulation of signaling; however, two variants behaved as the wild-type ACVRL1 did, while computational classifiers predicted their functional abnormalities. Three-dimensional structure prediction using the ColabFold program supported the significance of the L45 loop and NANDOR domain of ACVRL1 for its association with SMAD1 and BMPR2, respectively, and the variations in these motifs resulted in the reduction of SMAD signaling. On the other hand, two of the GS domain variants maintained high signal transduction capacity, which did not accord with their computational pathogenicity prediction. These results affirm the requirement of a combinatory approach using computational and experimental analyses to accurately predict the pathogenicity of ACVRL1 missense variants in the HHT patients.

The diverse effects of transforming growth factor-β and SMAD signaling pathways during the CTL response.

Cytotoxic T lymphocytes (CTLs) play an important role in defense against infections with intracellular pathogens and anti-tumor immunity. Efficient migration is required to locate and destroy infected cells in different regions of the body. CTLs accomplish this task by differentiating into specialized subsets of effector and memory CD8 T cells that traffic to different tissues. Transforming growth factor-beta (TGFβ) belongs to a large family of growth factors that elicit diverse cellular responses via canonical and non-canonical signaling pathways. Canonical SMAD-dependent signaling pathways are required to coordinate changes in homing receptor expression as CTLs traffic between different tissues. In this review, we discuss the various ways that TGFβ and SMAD-dependent signaling pathways shape the cellular immune response and transcriptional programming of newly activated CTLs. As protective immunity requires access to the circulation, emphasis is placed on cellular processes that are required for cell-migration through the vasculature.

The lncRNA LETS1 promotes TGF-β-induced EMT and cancer cell migration by transcriptionally activating a TβR1-stabilizing mechanism.

Transforming growth factor-β (TGF-β) signaling is a critical driver of epithelial-to-mesenchymal transition (EMT) and cancer progression. In SMAD-dependent TGF-β signaling, activation of the TGF-β receptor complex stimulates the phosphorylation of the intracellular receptor-associated SMADs (SMAD2 and SMAD3), which translocate to the nucleus to promote target gene expression. SMAD7 inhibits signaling through the pathway by promoting the polyubiquitination of the TGF-β type I receptor (TβRI). We identified an unannotated nuclear long noncoding RNA (lncRNA) that we designated LETS1 (lncRNA enforcing TGF-β signaling 1) that was not only increased but also perpetuated by TGF-β signaling. Loss of LETS1 attenuated TGF-β-induced EMT and migration in breast and lung cancer cells in vitro and extravasation of the cells in a zebrafish xenograft model. LETS1 potentiated TGF-β-SMAD signaling by stabilizing cell surface TβRI, thereby forming a positive feedback loop. Specifically, LETS1 inhibited TβRI polyubiquitination by binding to nuclear factor of activated T cells (NFAT5) and inducing the expression of the gene encoding the orphan nuclear receptor 4A1 (NR4A1), a component of a destruction complex for SMAD7. Overall, our findings characterize LETS1 as an EMT-promoting lncRNA that potentiates signaling through TGF-β receptor complexes.

TGF-β mediated drug resistance in solid cancer

Transforming growth factor β (TGF-β) is an important signaling molecule which is expressed in three different isoforms in mammals (i.e. TGF-β1, -β2, and -β3). The interaction between TGF-β and its receptor triggers several pathways, which are classified into SMAD-dependent (canonical) and SMAD-independent (non-canonical) signaling, whose activation/transduction is finely regulated by several mechanisms. TGF-β is involved in many physiological and pathological processes, assuming a dualistic role in cancer progression depending on tumor stage. Indeed, TGF-β inhibits cell proliferation in early-stage tumor cells, while it promotes cancer progression and invasion in advanced tumors, where high levels of TGF-β have been reported in both tumor and stromal cells. In particular, TGF-β signaling has been found to be strongly activated in cancers after treatment with chemotherapeutic agents and radiotherapy, resulting in the onset of drug resistance conditions. In this review we provide an up-to-date description of several mechanisms involved in TGF-β-mediated drug resistance, and we report different strategies that are currently under development in order to target TGF-β pathway and increase tumor sensitivity to therapy.

FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-β-Induced EMT

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial-mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.