Inhibition Of TGF-β Signaling Research Articles

How pancreatic adenocarcinoma might cause diabetes? The role of TGF- β

Pancreatic ductal adenocarcinoma (PDAC) is a deadly sickness that stays incurable due to past due diagnosis, which renders any healing intervention challenging. Most PDAC sufferers expand de novo diabetes, which exacerbates their morbidity and mortality. How PDAC triggers diabetes continues to be unfolding. Using a mouse version of KrasG12D-pushed PDAC, which faithfully recapitulates the development of the human sickness, we determined a large and selective depletion of β-cells, taking place very early on the degrees of preneoplastic lesions. Mechanistically, it turned into observed that accelerated TGF beta (TGF-β) signaling throughout PDAC development induced erosion of β-mobileular mass thru apoptosis. Suppressing TGF-β signaling, both pharmacologically thru TGF-β immunoneutralization or genetically thru deletion of Smad4 or TGF-β kind II receptor (TβRII), afforded size able safety in opposition to PDAC-pushed β-mobileular depletion. From a translational perspective, each activation of TGF-β signaling and depletion of β-cells often arise in human PDAC, imparting a mechanistic cause of the pathogenesis of diabetes in PDAC sufferers, and similarly implicating new- onset diabetes as a capability early prognostic marker for PDAC. In this mini review we try to analyze the principle relationships between pancreatic cancer and diabetes and vice versa in addition to the implication of TGF-β signaling as a likely goal for attenuating diabetes in pancreatic most cancers patients.

Abstract 14033: Inhibition of the Prolyl Isomerase Pin1 Improves Endothelial Function and Attenuates Vascular Remodelling in Pulmonary Hypertension by Inhibiting TGF-β Signalling

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-β/BMP signalling, endothelial cell dysfunction and vascular remodelling in PAH is unknown. Methods and Results: Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-β signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-β signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Conclusions: Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH-MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-β/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Inhibition of TGF-β Signaling Attenuates Disuse-induced Trabecular Bone Loss After Spinal Cord Injury in Male Mice.

Bone loss is one of the most common complications of immobilization after spinal cord injury (SCI). Whether transforming growth factor (TGF)-β signaling plays a role in SCI-induced disuse bone loss has not been determined. Thus, 16-week-old male mice underwent sham or spinal cord contusion injury to cause complete hindlimb paralysis. Five days later, 10 mg/kg/day control (IgG) or anti-TGF-β1,2,3 neutralizing antibody (1D11) was administered twice weekly for 4 weeks. Femurs were examined by micro-computed tomography (micro-CT) scanning and histology. Bone marrow (BM) supernatants were analyzed by enzyme-linked immunosorbent assay for levels of procollagen type 1 intact N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase (TRAcP-5b), receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and prostaglandin E2 (PGE2). Distal femoral micro-CT analysis showed that SCI-1D11 mice had significantly (P < .05) attenuated loss of trabecular fractional bone volume (123% SCI-1D11 vs 69% SCI-IgG), thickness (98% vs 81%), and connectivity (112% vs 69%) and improved the structure model index (2.1 vs 2.7). Histomorphometry analysis revealed that osteoclast numbers were lower in the SCI-IgG mice than in sham-IgG control. Biochemically, SCI-IgG mice had higher levels of P1NP and PGE2 but similar TRAcP-5b and RANKL/OPG ratio to the sham-IgG group. The SCI-1D11 group exhibited higher levels of P1NP but similar TRAcP-5b, RANKL/OPG ratio, and PGE2 to the sham-1D11 group. Furthermore, 1D11 treatment prevented SCI-induced hyperphosphorylation of tau protein in osteocytes, an event that destabilizes the cytoskeleton. Together, inhibition of TGF-β signaling after SCI protects trabecular bone integrity, likely by balancing bone remodeling, inhibiting PGE2 elevation, and preserving the osteocyte cytoskeleton.

The GPI-anchored protein CD109 protects hematopoietic progenitor cells from undergoing erythroid differentiation induced by TGF-β.

Although a glycosylphosphatidylinositol-anchored protein (GPI-AP) CD109 serves as a TGF-β co-receptor and inhibits TGF-β signaling in keratinocytes, the role of CD109 on hematopoietic stem progenitor cells (HSPCs) remains unknown. We studied the effect of CD109 knockout (KO) or knockdown (KD) on TF-1, a myeloid leukemia cell line that expresses CD109, and primary human HSPCs. CD109-KO or KD TF-1 cells underwent erythroid differentiation in the presence of TGF-β. CD109 was more abundantly expressed in hematopoietic stem cells (HSCs) than in multipotent progenitors and HSPCs of human bone marrow (BM) and cord blood but was not detected in mouse HSCs. Erythroid differentiation was induced by TGF-β to a greater extent in CD109-KD cord blood or iPS cell-derived megakaryocyte-erythrocyte progenitor cells (MEPs) than in wild-type MEPs. When we analyzed the phenotype of peripheral blood MEPs of patients with paroxysmal nocturnal hemoglobinuria who had both GPI(+) and GPI(-) CD34+ cells, the CD36 expression was more evident in CD109- MEPs than CD109+ MEPs. In summary, CD109 suppresses TGF-β signaling in HSPCs, and the lack of CD109 may increase the sensitivity of PIGA-mutated HSPCs to TGF-β, thus leading to the preferential commitment of erythroid progenitor cells to mature red blood cells in immune-mediated BM failure.

Hyperkyphosis is not dependent on bone mass and quality in the mouse model of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-β. Angiotensin II (ang-II) is involved in TGF-β activity and in bone mass regulation. Inhibition of TGF-β signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8±6.3 vs. 29.7±7.7μm), coupled with a reduction in type I (164.1±8.7 vs. 139.0±4.4) and increase in type III (86.5±10.2 vs. 140.4±5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0±420.6 vs. 655.6±239.5μm2) surrounded by increased area of connective tissue (58.17±6.52 vs. 105.0±44.54μm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

Targeting of Smad7 in Mesenchymal Cells Does Not Exacerbate Fibrosis During Experimental Chronic Pancreatitis.

Transforming growth factor-β (TGF-β)-mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-β signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-β signaling in this cell type in mice. A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis. Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice was only slightly increased, without statistical significance. Except for fibronectin, the expression of different extracellular matrix proteins and the numbers of fibroblasts and inflammatory cells were similar between Smad7-mutant and control mice. There was no clear evidence that the lack of Smad7 in pancreatic stellate cells plays a major role in experimental pancreatitis, at least in the mouse model investigated here.

Long-Acting Tumor-Activated Prodrug of a TGFβR Inhibitor.

Inhibition of TGFβ signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFβ receptor (TGFβR) inhibitors in cancer therapy. To restrict the activity of TGFβR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFβR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Implications of TGFβ Signaling and CDK Inhibition for the Treatment of Breast Cancer.

Simple SummarySmall molecules that inhibit cyclin dependent kinases (CDKs) have great potential for the treatment of breast cancer and have been implemented in the standard of care for some patients with metastatic disease. As the indications for CDK inhibitors continue to expand, and it is crucial to understand the mechanism of action of these drugs and treatment interactions with other targeted therapies. Accordingly, this review discusses subtype-specific systemic breast cancer treatment, the effects of signaling through transforming growth factor beta (TGFβ), and the unique potential for implementation of CDK inhibitor therapy.TGFβ signaling enacts tumor-suppressive functions in normal cells through promotion of several cell regulatory actions including cell-cycle control and apoptosis. Canonical TGFβ signaling proceeds through phosphorylation of the transcription factor, SMAD3, at the C-terminus of the protein. During oncogenic progression, this tumor suppressant phosphorylation of SMAD3 can be inhibited. Overexpression of cyclins D and E, and subsequent hyperactivation of cyclin-dependent kinases 2/4 (CDKs), are often observed in breast cancer, and have been associated with poor prognosis. The noncanonical phosphorylation of SMAD3 by CDKs 2 and 4 leads to the inhibition of tumor-suppressive function of SMAD3. As a result, CDK overactivation drives oncogenic progression, and can be targeted to improve clinical outcomes. This review focuses on breast cancer, and highlights advances in the understanding of CDK-mediated noncanonical SMAD3 phosphorylation. Specifically, the role of aberrant TGFβ signaling in oncogenic progression and treatment response will be examined to illustrate the potential for therapeutic discovery in the context of cyclins/CDKs and SMAD3.

Angiotensin II receptor blockers in modulation of transforming growth factor-beta effects on mitral valve myxomatous degeneration

Abstract Growing evidence supports role transforming growth factor-β (TGF-β) as a mediator of the myxomatous changes in the mitral valve prolapse. Recently, angiotensin II receptor blockers (ARB) emerged as a potentially effective inhibitor of TGF-β signaling. Treatment of valvular interstitial cells (VICs) with ARB resulted in effective inhibition of TGF-β-induced ECM expression. Our aim was to evaluate the effect of preoperative ARB therapy in modulation of transforming growth factor-β effects on mitral valve myxomatous degeneration. Methods A total of 233 asymptomatic patients (mean age: 53.8±12.9) undergoing mitral valve surgery for severe mitral regurgitation due to mitral valve prolapse were enrolled in our retrospective, non-randomized, single-center study. Resected abnormal segments of the mitral leaflets (segment of the posterior leaflet or the entire valve) were obtained during mitral valve repair/replacement surgery and examined by experienced pathologists. Immunohistochemical characterization of mitral valve sections was performed with the primary antibodies. Quantification was performed by counting TGF-β1 and TGF-β2 positive cells within 10 random high-power fields divided by the total number of cells in specimens. Quantification of immunohistochemical staining for collagen III was performed by measurement of staining area divided by the total area of the specimens. Results Two hundred thirty-three consecutive patients (66% males, mean age 53.8±12.9 years, range 19–80) with severe MR were enrolled in the study. According to the case reports, 43 patients (18.5%) received losartan or telmisartan before surgery and were included in study group. 190 patients (81.5%) did not receive any ARB and were enrolled in the control group. Histological examination showed disorganized collagen and elastin fibers, expansion of the spongiosa layer in both groups. However, MV specimens' immunohistochemistry revealed a lower expression of type III collagen in ARB group as compared to controls (31.3% ± 10.8% vs. 43.8% ± 15.3%; p&amp;lt;0.001). MV leaflets in control group showed the increased VICs density (95.3±39.9 vs. 70.0±21.7/high-power field, p=0.0001). TGF-β1 and TGF-β2 positive cells were significantly rare in the ARB than in control MV specimens (18% vs. 33%, p=0.012 and 16% vs. 38%, p&amp;lt;0.ehab724.15541, respectively). Expression of type III collagen and TGF-β2 positive cells count showed a weak, but significant correlation (r=0.28; p=0.ehab724.155414). The multivariate Cox analysis showed a decrease in the relative risk of valvular thickening (myxomatous degeneration) with ARB therapy (0.85; 95% CI: 0.61 – 1.17; p=0.01). Conclusions We indicate a beneficial effect of ARB treatment through the inhibition of TGF-β pathway on valvular myxomatous degeneration in patients with MVP. Modulation of the progression of MVP with such therapeutic agents may have great clinical significance. Funding Acknowledgement Type of funding sources: None.

P66.03 The Functional Role of TGF-β Signaling in SCLC Heterogeneity and Metastasis

Small cell lung cancer (SCLC) is the most aggressive lung cancer and highly heterogeneous phenotypes have been observed in human and mouse SCLC cells. Tumor heterogeneity has been proven to contribute to SCLC malignant progression and/or metastasis in RP-based models. And the unique therapeutic vulnerabilities across different SCLC subtypes have been linked with their specific gene expression profile, indicating the need of personal therapeutic strategy in clinic. The TGF-β signaling consists of secreted TGF-β family members, receptors and SMAD proteins, and is implicated in multiple cell biological processes, such as cell proliferation and survival, epithelial to mesenchymal transition (EMT), cell fate control, immune response, and tumorigenesis. Dysregulation of the TGF-β signaling pathway has been reported in different cancer types and dual role of this pathway in cancer development and progression has been documented. Activation of this pathway results in cell cycle arrest and apoptosis at the early-stage of cancer development, indicating a tumor suppressive function. On the other hand, TGF-β could promote EMT as well as tumor malignant progression at the late-stage disease. Up to date, the contribution of the TGF-β pathway in SCLC development and progression remains elusive. Using the Rb1L/L/Trp53 L/L (RP) mouse model, we find that mouse SCLC cells with adherent growth pattern are composed of mesenchymal-like (Mes) and epithelial-like (Epi) subpopulations. Take advantage of in vitro functional assay and in vivo allograft assay, we find that the Mes cells harbor higher malignant transformation and metastatic capabilities than Epi cells. Through the analysis of microarray data, we find that the TGF-β signaling is enriched in Mes cells. And the inhibition of TGF-β signaling in Mes cells significantly abrogates its metastatic ability in allograft assay. Moreover, genetic deletion of the key components of the TGF-β signaling pathway, dramatically attenuated SCLC metastasis in the RP mouse model. In this study, we find that mouse SCLC cells derived from RP autochthonous mouse model with adherent growth pattern are composed of Mes and Epi subpopulations. The Mes cells have increased ability to form colonies in soft agar and harbored stronger metastatic capability in vivo when compared to the Epi cells. Gene Set Enrichment Analysis (GSEA) reveals that the TGF-β signaling is enriched in the Mes cells. Inhibition of the TGF-β signaling through either ectopic expression of dominant negative Tgfbr2 (Tgfbr2-DN) or treatment with Tgfbr1 inhibitor SD-208 consistently greatly abrogates the tumor metastasis in allograft assays. Moreover, genetic deletion of Tgfbr2 or Smad4, key components of the TGF-β signaling pathway, dramatically attenuates SCLC metastasis in the RP autochthonous mouse model of SCLC. Our results suggest the critical role of TGF-β signaling in SCLC heterogeneity and metastasis. Importantly, inhibition of TGF-β signaling significantly abrogates SCLC metastasis, providing a potential therapeutic avenue for SCLC management in clinic.

Tetraethylthiuram disulphide alleviates pulmonary fibrosis through modulating transforming growth factor-β signalling

Idiopathic pulmonary fibrosis (IPF) induces significant morbidity and mortality, for which there are limited therapeutic options available. Here, we found that tetraethylthiuram disulphide (disulfiram, DSF), a derivative of thiuram, used in the treatment of alcohol abuse, has an inhibitory effect on bleomycin (BLM)-induced pulmonary fibrosis via the attenuation of the fibroblast-to-myofibroblast transition, migration, and proliferation of fibroblasts. Furthermore, DSF inhibited the activation of primary pulmonary fibroblasts and fibroblast cell line under transforming growth factor-β 1 (TGF-β1) challenge. Mechanistically, the anti-fibrotic effect of DSF on fibroblasts depends on the inhibition of TGF-β signalling. We further determined that DSF interrupts the interaction between SMAD3 and TGF-β receptor Ι (TBR Ι), and identified that DSF directly binds with SMAD3, in which Trp326, Thr330, and Cys332 of SMAD3 are critical binding sites for DSF. Collectively, our results reveal a powerful anti-fibrotic function of DSF in pulmonary fibrosis through the inhibition of TGF-β/SMAD signalling in pulmonary fibroblasts, indicating that DSF is a promising therapeutic candidate for IPF.

Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuron–stimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFβ signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.

Tumor-derived MMPs regulate cachexia in a Drosophila cancer model.

Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. We have established a Drosophila larval model of organ wasting whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGFβ signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFβ signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, our study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia.

MiR-216a-5p alleviates LPS-induced inflammation in the human bronchial epithelial cell by inhibition of TGF-β1 signaling via down-regulating TGFBR2.

Bronchopneumonia is a common respiratory infection disease and is the leading cause of hospitalization in children under 5 years of age. Inflammation is the primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation induced by bronchopneumonia and investigate the potential mechanism underlying it. Human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccha-rides (LPS) to trigger bronchopneumonia in vitro. The production of interleukin (IL)-1β, IL-6, and Tumor necrosis factor (TNF)-α was measured using the enzyme-linked immunosorbent assay. The luciferase assay was conducted to explore the relationship between miR-216a-5p and TGFBR2. Quantitative real-time polymerase chain reaction and western blot were used to detect the gene expression. miR-216a-5p gene expression decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of IL-1β, IL-6, and TNF-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor (TGF)-β1, and phosphorylation of SMAD family member 2 (smad2),. This ectopic expression of miR-216a-5p was restored by overexpressed TGFBR2. miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-β1 signaling via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia.Bronchopneumonia is a common respiratory infection disease and is the main cause of hospitalization in children under 5 years of age. Inflammation is a primary response caused by bronchopneumonia. But the detailed underlying mechanism of inflammation in bronchopneumonia remains unclear. Therefore, this study focused on studying the effect of miR-216a-5p on inflammation caused by bronchopneumonia and investigate the potential mechanism underlying it. In this study, human bronchial epithelial cells (BEAS-2B) were stimulated using lipopolysaccharides (LPS) to trigger bronchopneumonia in vitro. miR-216a-5p was decreased in BEAS-2B cells stimulated by LPS. Overexpression of miR-216a-5p suppressed the elevated levels of interleukin (IL)-1β, IL-6, and Tumor necrosis factor (TNF)-α induced by LPS. Transforming growth factor-beta receptor 2 (TGFBR2) proved to be a direct target of miR-216a-5p, and they negatively modulated TGFBR2 expression. In addition, overexpression of miR-216a-5p inhibited LPS-induced protein levels of TGFBR2,transforming growth factor-beta 1 (TGF-β1), and phosphorylation of SMAD family member 2 (smad2. This ectopic overexpression of miR-216a-5p was restored by overexpressed TGFBR2. In conclusion, miR-216a-5p was decreased in LPS-stimulated BEAS-2B cells. Overexpressed miR-216a-5p suppressed LPS-induced inflammation in BEAS-2B cells by inhibition of TGF-β1 signaling via down-regulating TGFBR2. miR-216a-5p may be a valuable target for anti-inflammation treatment in bronchopneumonia.

Abstract MP24: Disruption Of Actin Remodeling Intensifies Aneurysm-promoting Signals In Smooth Muscle Cells

Mutations within proteins coding for extracellular matrix assembly or those in the transforming growth factor beta (TGFβ) signaling pathway lead to aneurysm formation in patients and mice. As the SMC cytoskeletal network is intricately linked to TGFβ and ECM signaling, we sought to determine if disruption of the cytoskeletal filaments would alter how SMCs respond to angiotensin II, an important mediator of aneurysm formation. C57BL/6 mice lacking Tgbfr1 in SMCs (SMC- Tgbr1 iko ) generated by Jiang et al. have been shown to develop rapid and severe aortic aneurysm degeneration with 100% penetrance. Analysis was conducted on aneurysm and control mouse and human aortic tissue sections. We observed that SMC- Tgbr1 iko mice had reduced expression of filamentous (f-) and α-actin protein in the aortic media than control mice. Primary Tgbr1 iko SMCs also had higher levels of pRelA expression, less α-actin mRNA, and less f-actin than control SMCs. Tissue sections from aneurysm patients had reduced f-actin staining relative to controls. Disruption of f-actin with the destabilizer latrunculin A in vitro led to increased RelA and ERK activation in the presence of AngII, which was further augmented in the presence of a neutralizing antibody for TGFβ. In contrast, jasplakinolide—an f-actin stabilizer—prevented TGFβ blockade induced pRelA expression, suggesting that altered/inhibited TGFβ signaling modulates NFkB through actin depolymerization. While stabilization and disruption of microtubules led to RelA activation, no effect was observed on TGFβ blockade-induced pRelA expression. The phosphoantibody array revealed that f-actin disruption in the presence of AngII activated the CREB, AKT1/2/3, ERK1/2, and STAT3 pathways in mouse aortic SMCs. SMC- Tgbr1 iko mice treated with cytochalasin B suffered significantly greater mortality and had increased aortic growth over a 30-day follow-up period. Aortic aneurysms are coincident with reduced f-actin expression in murine and human aortic SMCs. Chemical deconstruction of actin filaments promotes an inflammatory phenotype in mouse SMCs in vitro , and aggravates aneurysm formation in vivo . Aberrant or inhibited TGFβ signaling potentiates NFkB activation in SMCs, which may be dependent on f-actin disassembly.

TGFβ signalling is required to maintain pluripotency of human naïve pluripotent stem cells.

The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFβ signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naive to primed states.

Curcumin Inhibition of TGFβ signaling in bone metastatic breast cancer cells and the possible role of oxidative metabolites

TGFβ signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFβ signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFβ signaling and the application of this finding across multiple BCa cell lines forming TGFβ-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFβ signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFβ-induced Smad activation due to down-regulation of plasma membrane associated TGFβR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFβR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFβ signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFβR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFβ-dependent BCa BMETs.

Kurarinone Attenuates BLM-Induced Pulmonary Fibrosis via Inhibiting TGF-β Signaling Pathways.

Idiopathic pulmonary fibrosis (IPF) is a refractory interstitial lung disease for which there is no effective treatment. Although the pathogenesis of IPF is not fully understood, TGF-β and epithelial–mesenchymal transition (EMT) have been shown to be involved in the fibrotic changes of lung tissues. Kurarinone is a prenylated flavonoid isolated from Sophora Flavescens with antioxidant and anti-inflammatory properties. In this study, we investigated the effect of kurarinone on pulmonary fibrosis. Kurarinone suppressed the TGF-β-induced EMT of lung epithelial cells. To assess the therapeutic effects of kurarinone in bleomycin (BLM)-induced pulmonary fibrosis, mice were treated with kurarinone daily for 2 weeks starting 7 days after BLM instillation. Oral administration of kurarinone attenuated the fibrotic changes of lung tissues, including accumulation of collagen and improved mechanical pulmonary functions. Mechanistically, kurarinone suppressed phosphorylation of Smad2/3 and AKT induced by TGF-β1 in lung epithelial cells, as well as in lung tissues treated with BLM. Taken together, these results suggest that kurarinone has a therapeutic effect on pulmonary fibrosis via suppressing TGF-β signaling pathways and may be a novel drug candidate for pulmonary fibrosis.

Protective function of interleukin-22 in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive scarring disease with unknown etiology. The evidence of a pathogenic role for transforming growth factor‐beta (TGF‐β) in the development and progression of IPF is overwhelming. In the present study, we investigated the role of interleukin‐22 (IL‐22) in the pathogenesis of IPF by regulating the TGF‐β pathway. We measured parameters and tissue samples from a clinical cohort of IPF. IL‐22R knock out (IL‐22RA1−/−) and IL‐22 supplementation mouse models were used to determine if IL‐22 is protective in vivo. For the mechanistic study, we tested A549, primary mouse type II alveolar epithelial cell, human embryonic lung fibroblast, and primary fibroblast for their responses to IL‐22 and/or TGF‐β1. In a clinical cohort, the expression level of IL‐22 in the peripheral blood and lung tissues of IPF patients was lower than healthy controls, and the lower IL‐22 expression was associated with poorer pulmonary function. IL‐22R−/− mice demonstrated exacerbated inflammation and fibrosis. Reciprocally, IL‐22 augmentation by intranasal instillation of recombinant IL‐22 repressed inflammation and fibrotic phenotype. In vitro, IL‐22 treatment repressed TGF‐β1 induced gene markers representing epithelial‐mesenchymal‐transition and fibroblast‐myofibroblast‐transition, likely via the inhibition of TGF‐β receptor expression and subsequent Smad2/3 activation. IL‐22 appears to be protective against pulmonary fibrosis by inhibiting TGF‐β1 signaling, and IL‐22 augmentation may be a promising approach to treat IPF.

Osteoarthritis-Related Inflammation Blocks TGF-β's Protective Effect on Chondrocyte Hypertrophy via (de)Phosphorylation of the SMAD2/3 Linker Region.

Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-β (TGF-β) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-β signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1β and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-β signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1β was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.