Perturbation of transforming growth factor (TGF)-ß1 association with latent TGF-β binding protein yields inflammation and tumors

Transforming growth factor-β (TGF-β) activity is controlled at many levels including the conversion of the latent secreted form to its active state. TGF-β is often released as part of an inactive tripartite complex consisting of TGF-β, the TGF-β propeptide, and a molecule of latent TGF-β binding protein (LTBP). The interaction of TGF-β and its cleaved propeptide renders the growth factor latent, and the liberation of TGF-β from this state is crucial for signaling. To examine the contribution of LTBP to TGF-β function, we generated mice in which the cysteines that link the propeptide to LTBP were mutated to serines, thereby blocking covalent association. Tgfb1^C33S/C33S mice had multiorgan inflammation, lack of skin Langerhans cells (LC), and a shortened lifespan, consistent with decreased TGF-β1 levels. However, the inflammatory response and decreased lifespan were not as severe as observed with Tgfb1^−/− animals. Tgfb1^C33S/C33S mice exhibited decreased levels of active TGF-β1, decreased TGF-β signaling, and tumors of the stomach, rectum, and anus. These data suggest that the association of LTBP with the latent TGF-β complex is important for proper TGF-β1 function and that Tgfb1^C33S/C33S mice are hypomorphs for active TGF-β1. Moreover, although mechanisms exist to activate latent TGF-β1 in the absence of LTBP, these mechanisms are not as efficient as those that use the latent complex containing LTBP.     

Linker phosphorylation of Smad3 promotes fibro-carcinogenesis in chronic viral hepatitis of hepatocellular carcinoma

Epidemiological and clinical data point to a close association between chronic hepatitis B virus infection or chronic hepatitis C virus infection and development of hepatocellular carcinoma (HCC). HCC develops over several decades and is associated with fibrosis. This sequence suggests that persistent viral infection and chronic inflammation can synergistically induce liver fibrosis and hepatocarcinogenesis. The transforming growth factor-β (TGF-β) signaling pathway plays a pivotal role in diverse cellular processes and contributes to hepatic fibro-carcinogenesis under inflammatory microenvironments during chronic liver diseases. The biological activities of TGF-β are initiated by the binding of the ligand to TGF-β receptors, which phosphorylate Smad proteins. TGF-β type I receptor activates Smad3 to create COOH-terminally phosphorylated Smad3 (pSmad3C), while pro-inflammatory cytokine-activated kinases phosphorylates Smad3 to create the linker phosphorylated Smad3 (pSmad3L). During chronic liver disease progression, virus components, together with pro-inflammatory cytokines and somatic mutations, convert the Smad3 signal from tumor-suppressive pSmad3C to fibro-carcinogenic pSmad3L pathways, accelerating liver fibrosis and increasing the risk of HCC. The understanding of Smad3 phosphorylation profiles may provide new opportunities for effective chemoprevention and personalized therapy for patients with hepatitis virus-related HCC in the future.     

Stromal growth and epithelial cell proliferation in ventral prostates of liver X receptor knockout mice

With specific liver X receptor α and β (LXRα and LXRβ) antibodies, we found that LXRα is strongly expressed in the luminal and basal cells of prostatic epithelium. The ventral prostates (VP) of LXRα^−/− mice are characterized by the presence of smooth-muscle actin-positive stromal overgrowth around the prostatic ducts and by numerous fibrous nodules pushing into the ducts and causing obstruction, so that most of the ducts were extremely dilated. BrdU labeling and Ki67 staining revealed epithelial and stromal proliferation in the fibrous nodules. However, the dense stroma surrounding the ducts was not positive for proliferation markers. There was no detectable difference between WT and LXRα^−/− mice VP in the expression of the androgen receptor, but there was an increase in nuclear expression of Snail and Smad 2/3, indicating enhanced TGF-β signaling. Upon treatment of WT mice for 3 months with the LXR agonist T2320 or for 3 weeks with β-sitosterol, LXRα was downregulated, and a VP phenotype similar to that of LXRα^−/− mice resulted. We conclude that in rodents, LXRα seems to control VP stromal growth and that LXRα^−/− mice may be a useful model to study prostatic stromal hyperplasia. Because LXRα is expressed in the epithelium, the excessive stromal growth in LXRα^−/− mice indicates that LXRα is essential for epithelial stromal communication.     

Inhibitors of Activin Receptor-like Kinase 5 Interfere with SARS-CoV-2 S-Protein Processing and Spike-Mediated Cell Fusion via Attenuation of Furin Expression

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor β (TGF-β) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-β1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-β1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell–cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell–cell spread via downregulation of furin which is most pronounced upon TGF-β stimulation. Since a TGF-β dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.     

Association between TGF-β gene polymorphism and myopia: A systematic review and meta-analysis

Introduction:The present study was conducted to determine the association of transforming growth factor-beta (TGF-β) gene polymorphism and myopia.Method:Four hundred twelve articles were identified, of which 11 articles with 5213 participants in 4 countries were included in the final analysis. Review Manager software (RevMan, version 5.4) was used for data analysis.Result:Odds ratio (OR) value of TGF-β1 rs1800469 is 1.33 (95% confidence interval [CI] = 1.15–1.54) in the allelic model; in the dominant model is 1.76 (95% CI = 1.16–2.67); in homozygous model is 5.98 (95% CI = 4.31–8.06). OR value of TGF-β1 rs4803455 is 0.62 (95% CI = 0.43–0.88) in recessive model. TGF-β2 is not associated with myopia. Relevant study on TGF-β3 is scarce.Conclusion:Our systematic review and meta-analysis found that TGF-β1 rs4803455 and rs1800469 were correlated with myopia.     

Identification of amino acids essential for the antiangiogenic activity of tumstatin and its use in combination antitumor activity

Tumstatin is an angiogenesis inhibitor that binds to αvβ3 integrin and suppresses tumor growth. Previous deletion mutagenesis studies identified a 25-aa fragment of tumstatin (tumstatin peptide) with in vitro antiangiogenic activity. Here, we demonstrate that systemic administration of this tumstatin peptide inhibits tumor growth and angiogenesis. Site-directed mutagenesis identified amino acids L, V, and D as essential for the antiangiogenic activity of tumstatin. The tumstatin peptide binds to αvβ3 integrin on proliferating endothelial cells and localizes to select tumor endothelium in vivo. Using 3D molecular modeling, we identify a putative interaction interface for tumstatin peptide on αvβ3 integrin. The antitumor activity of the tumstatin peptide, in combination with bevacizumab (anti-VEGF antibody), displays significant improvement in efficacy against human renal cell carcinoma xenografts when compared with the single-agent use. Collectively, our results demonstrate that tumstatin peptide binds specifically to the tumor endothelium, and its antiangiogenic action is mediated by αvβ3 integrin, and, in combination with an anti-VEGF antibody it exhibits enhanced tumor suppression of renal cell carcinoma.     

Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β₂-adrenergic receptors (β₂AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β₂AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β₂AR actions favorable for treating obstructive lung disease.

Most G protein–coupled receptors (GPCRs) are now recognized as multisignal transducers (1, 2). Early concepts of agonist–receptor interactions were based on the idea that there was a single “active” receptor conformation induced by the binding of any agonist, resulting in an interaction with the heterotrimeric G protein and a universal, singular signal. Generally, the α-subunit of the G protein, upon its dissociation, was considered the primary activator (or inhibitor) of the effector, resulting in the intracellular signal. Subsequently, it became clear that multiple signaling outcomes from activation of a given GPCR can occur from a single agonist due to specific molecular determinants of the receptor triggering independent mechanisms (3–5). As these multiple functions were being identified, it was apparent that agonists with different structures could act at a given receptor to preferentially activate one signal with minimal engagement of others, a property later termed signal biasing (6–8). Biased agonists, then, could represent important advantages over nonbiased agonists due to this signal selectivity, activating a specified therapeutic pathway while minimally evoking unnecessary or deleterious signaling. The pathway selectivity of biased agonists is thought to be established by the stabilization of specific conformation(s) of the agonist–receptor complex via a set of interactions that differ from those of unbiased (also called balanced) agonists (9–12). While it is conceivable that small modifications of established cognate agonists might yield such specialized signaling, significant deviation from common agonist structures may be necessary to meet this goal (13).The signals/functions of a given GPCR that might be sought for selective activation are defined by the cell type, disease, and desired final physiologic function. In asthma and chronic obstructive pulmonary disease (COPD), active human airway smooth muscle (HASM) cellular contraction limits airflow, representing a major cause of morbidity and mortality. β₂-adrenergic receptors (β₂ARs) expressed on HASM cells are the targets for binding of therapeutically administered β-agonists, which relax the cells via a cyclic adenosine monophosphate–mediated mechanism (14). β-agonists are used for treating acute bronchospasm as well as for long-term prevention. However, the HASM bronchodilator response to acute β-agonist is attenuated by receptor desensitization (15), with typical treatments of humans, or isolated HASM cells, leading to a loss of receptor function over time (16–18), clinically termed tachyphylaxis.Agonist-promoted desensitization of β₂AR (and other GPCRs) is due to partial uncoupling of the receptor to the G protein, which is initiated by phosphorylation of intracellular Ser/Thr residues of the receptor by G protein–coupled receptor kinases (GRKs) (19, 20). The GRK-phosphorylated β₂AR recruits β-arrestin1 or β-arrestin2 to these receptors, with subsequent interactions that appear to compete with the receptor for its binding to the Gα subunit, thus attenuating the intracellular response (11, 21). Such competition has been strongly inferred for the β₂AR (22, 23) and is compelling for rhodopsin–arrestin interactions (24). In addition, β-arrestin binding to GPCRs can initiate receptor internalization and other events such as receptor activation of ERK1/2 (25) through its multiprotein adapter functions. Thus β-arrestin engagement can be considered an early “second signal” of the β₂AR as well as a desensitization initiator for attenuating the Gs signal. An agonist that is biased toward Gαs coupling (cAMP production and airway smooth muscle [ASM] relaxation) and away from β-arrestin binding (desensitization) would be desirable in treating obstructive lung diseases, since efficacy would not be attenuated acutely, nor would tachyphylaxis be experienced from extended treatment. While biased agonists favoring either G protein or β-arrestin (6) signaling have been described for some GPCRs (such as μ-opioid and type 1 angiotensin II receptors), Gαs biasing has not been apparent from most studies with catecholamine-like compounds for the β₂AR. Thus, we have little information as to whether the two β₂AR pathways can be differentially activated in a selective manner by an efficacious agonist, nor is it apparent from a structural standpoint what strategy might be employed to design agonists biased in this manner for this receptor.In order to find this type of biasing for the β₂AR, we screened a 40-million-compound scaffold ranking (SR) library that was agnostic to known β₂AR agonist structures. We found a scaffold in which substitutions of certain R groups led to individual compounds that are apparently Gαs-biased agonists for β₂AR with no apparent engagement of β-arrestin in model systems. Additional studies in HASM cells revealed a lack of tachyphylaxis of the relaxation effect by the lead compound compared with the most widely utilized β₂AR agonist, albuterol. The structure of this biased agonist is very different from that of catecholamine-like agonists. To ascertain the mechanism that may underlie this biased activity, we used structural modeling and molecular simulations and studied homologous compounds with different R groups and receptor mutagenesis to predict the interaction sites with the activated β₂AR. Such studies uncovered distinct structural characteristics that may be responsible for the biasing effect.     

Inhibition of miR-155 Promotes TGF-β Mediated Suppression of HIV Release in the Cervical Epithelial Cells

TGF-β has been shown to play a differential role in either restricting or aiding HIV infection in different cell types, however its role in the cervical cells is hitherto undefined. Among females, more than 80% of infections occur through heterosexual contact where cervicovaginal mucosa plays a critical role, however the early events during the establishment of infection at female genital mucosa are poorly understood. We earlier showed that increased TGF-β level has been associated with cervical viral shedding in the HIV infected women, however a causal relationship could not be examined. Therefore, here we first established an in vitro cell-associated model of HIV infection in the cervical epithelial cells (ME-180) and demonstrated that TGF-β plays an important role as a negative regulator of HIV release in the infected cervical epithelial cells. Inhibition of miR-155 upregulated TGF-β signaling and mRNA expression of host restriction factors such as APOBEC-3G, IFI-16 and IFITM-3, while decreased the HIV release in ME-180 cells. To conclude, this is the first study to decipher the complex interplay between TGF-β, miR-155 and HIV release in the cervical epithelial cells. Collectively, our data suggest the plausible role of TGF-β in promoting HIV latency in cervical epithelial cells which needs further investigations.     

Getting ‘Smad' about obesity and diabetes

Recent findings on the role of transforming growth factor (TGF)-β/Smad3 signaling in the pathogenesis of obesity and type 2 diabetes have underscored its importance in metabolism and adiposity. Indeed, elevated TGF-β has been previously reported in human adipose tissue during morbid obesity and diabetic neuropathy. In this review, we discuss the pleiotropic effects of TGF-β/Smad3 signaling on metabolism and energy homeostasis, all of which has an important part in the etiology and progression of obesity-linked diabetes; these include adipocyte differentiation, white to brown fat phenotypic transition, glucose and lipid metabolism, pancreatic function, insulin signaling, adipocytokine secretion, inflammation and reactive oxygen species production. We summarize the recent in vivo findings on the role of TGF-β/Smad3 signaling in metabolism based on the studies using Smad3^−/− mice. Based on the presence of a dual regulatory effect of Smad3 on peroxisome proliferator-activated receptor (PPAR)β/δ and PPARγ2 promoters, we propose a unifying mechanism by which this signaling pathway contributes to obesity and its associated diabetes. We also discuss how the inhibition of this signaling pathway has been implicated in the amelioration of many facets of metabolic syndromes, thereby offering novel therapeutic avenues for these metabolic conditions.     

Transforming Growth Factor-β1 and Receptor for Advanced Glycation End Products Gene Expression and Protein Levels in Adolescents with Type 1 Diabetes Mellitus

Objective:Type 1 diabetes (T1D) mellitus is one of the most frequent autoimmune diseases in childhood. Chronic complications are the main causes of cardiovascular morbidity and mortality in T1D. Although interactions between advanced glycation end products (AGE) and their receptors (RAGE) and transforming growth factor-β1 (TGF-β1) are implicated in development and progression of diabetic microand macro-vascular complications, they also have important roles in immune system regulation.Methods:Blood samples were obtained from 156 adolescents with T1D and 80 apparently healthy controls. T1D patients diagnosed with any other autoimmune disease and receiving any kind of drugs except insulin therapy were excluded from this study. Exclusion criteria for controls were positive family history of T1D and drugs/supplements application. TGF-β1 and transmembrane full-length RAGE (flRAGE) messenger ribonucleic acid (mRNA) levels in peripheral blood mononuclear cells (PBMC) were obtained by quantitative polymerase chain reaction (qPCR) method. Circulating levels of biochemical markers, TGF-β1 and soluble RAGE (sRAGE) levels were also determined.Results:TGF-β1 and flRAGE mRNA levels were significantly higher in controls compared to patients (p<0.001, for both). However, TGF-β1 and sRAGE levels were higher in patients than controls (p<0.001, for both). There were significant independent associations of all mRNA and protein levels with T1D. TGF-β1 mRNA was the only marker independently negatively associated with urinary albumin excretion rate in T1D adolescents (p=0.005).Conclusion:Our results indicated gene expression downregulation of TGF-β1 and flRAGE in PBMC of T1D adolescents. TGF-β1 mRNA downregulation may be useful for predicting early elevation of urinary albumin excretion rate.     

Compromised peroxisomes in idiopathic pulmonary fibrosis,a vicious cycle inducing a higher fibrotic response via TGF-β signaling

Idiopathic pulmonary fibrosis (IPF) is a devastating disease, and its pathogenic mechanisms remain incompletely understood. Peroxisomes are known to be important in ROS and proinflammatory lipid degradation, and their deficiency induces liver fibrosis. However, altered peroxisome functions in IPF pathogenesis have never been investigated. By comparing peroxisome-related protein and gene expression in lung tissue and isolated lung fibroblasts between human control and IPF patients, we found that IPF lungs exhibited a significant down-regulation of peroxisomal biogenesis and metabolism (e.g., PEX13p and acyl-CoA oxidase 1). Moreover, in vivo the bleomycin-induced down-regulation of peroxisomes was abrogated in transforming growth factor beta (TGF-β) receptor II knockout mice indicating a role for TGF-β signaling in the regulation of peroxisomes. Furthermore, in vitro treatment of IPF fibroblasts with the profibrotic factors TGF-β1 or tumor necrosis factor alpha (TNF-α) was found to down-regulate peroxisomes via the AP-1 signaling pathway. Therefore, the molecular mechanisms by which reduced peroxisomal functions contribute to enhanced fibrosis were further studied. Direct down-regulation of PEX13 by RNAi induced the activation of Smad-dependent TGF-β signaling accompanied by increased ROS production and resulted in the release of cytokines (e.g., IL-6, TGF-β) and excessive production of collagen I and III. In contrast, treatment of fibroblasts with ciprofibrate or WY14643, PPAR-α activators, led to peroxisome proliferation and reduced the TGF-β–induced myofibroblast differentiation and collagen protein in IPF cells. Taken together, our findings suggest that compromised peroxisome activity might play an important role in the molecular pathogenesis of IPF and fibrosis progression, possibly by exacerbating pulmonary inflammation and intensifying the fibrotic response in the patients.Idiopathic pulmonary fibrosis (IPF) is a chronic, devastating, and lethal fibrotic disorder in human lung. IPF is characterized by a worsening of pulmonary function and persistent alterations of the lung parenchyma as a result of fibrotic foci formation by activated fibroblasts/myofibroblasts and excessive production and deposition of extracellular matrix components (ECM) (1–4). It is well accepted that transforming growth factor beta (TGF-β) signaling plays a critical role in IPF development. Inhibition of TGF-β signaling by blocking its downstream Smad3 gene expression protects against bleomycin-induced fibrosis in animal models (5, 6). In addition, there is increasing evidence that tumor necrosis factor alpha (TNF-α) also plays an important role in initiation and perpetuation of the fibrotic processes, possibly by activating TGF-β signaling pathway (7). However, the mechanisms by which TGF-β and TNF-α promote the fibrotic response in IPF are incompletely known.Peroxisomes are single membrane bounded ubiquitous organelles, present in all types of cells. Particularly, type II alveolar epithelial cells and club cells (Clara) in the lung have highly abundant peroxisomes (8). These organelles are involved in a variety of metabolic pathways, including degradation of reactive oxygen species (ROS) and bioactive lipid mediators (prostaglandins and leukotriens) and synthesis of antioxidant lipids (polyunsaturated fatty acids, plasmalogens, etc.) (9). Absence or dysfunction of peroxisomes results in increased cellular oxidative stress, leading to severe pathological consequences in many organ systems (10, 11). Lung is one of the organs with highest exposure to various forms of reactive oxygen and nitrogen species (ROS and RNS) due to oxygen and different environmental oxidants in the inspired air, causing oxidation of cellular DNA, proteins and lipids, consequently a direct lung injury (12). Studies have shown that the most severe phenotype of a peroxisome biogenesis disorder (e.g., Zellweger syndrome) is associated with progressive liver fibrosis or cirrhosis, leading to early death of the patients during childhood (11). Moreover, mice with peroxisome dysfunction caused by PEX11β knockout died during their first days of life and exhibit morphological alterations of the lungs (13). In contrast, treatment of rats with an agonist specific for peroxisome proliferator-activated receptor alpha (PPAR-α) significantly ameliorated tubulointerstitial renal fibrosis (14). Despite the fact that peroxisomal metabolism might play an important role in other tissue fibrosis, the role of peroxisomes in lung fibrosis onset and progression seen in IPF patients has never been reported (1, 15).Herein, using human IPF and control fibroblast cultures as well as a bleomycin-induced mouse lung fibrosis model, we demonstrate that peroxisomal biogenesis and metabolism is compromised in the lung and in fibroblasts of IPF patients, in which a down-regulation of peroxisomal proteins leads to activation and release of profibrotic factors such as TGF-β1 and collagen. In contrast, peroxisome proliferation by treatment with PPAR-α agonist (ciprofibrate, WY14643) significantly reduces the TGF-β1–induced myofibroblast differentiation in IPF fibroblast cultures.     

Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1β1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2β3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2β3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO₄ oxidation of the third digitoxose and reductive amination with various R-NH₂ substituents and identified derivatives with significant selectivity for human α2β1 over α1β1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the β1 subunit, Gln84, suggesting a role for β in digoxin binding. Gln84 in β1 is replaced by Val88 in β3. Assuming that alkyl substituents might interact with β3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2β3 over α1β1 (22–33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2β3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.Glaucoma is a disease leading to irreversible blindness. Control of intraocular pressure (IOP) is the mainstay of glaucoma therapy and is achieved by various drugs, such as β-blockers, prostaglandin analogs, α2 adrenergic receptor agonists, cholinergic agonists, and carbonic anhydrase inhibitors given topically or systemically (1). The topical route minimizes systemic side effects and is preferable, provided the drug effectively permeates the cornea. Despite the selection of drugs available, uncontrolled IOP in many patients eventually makes surgical intervention necessary. Thus, fresh approaches to drug treatments are required.The Na,K-ATPase provides the motive power for aqueous humor production in the ciliary epithelium. The sodium, potassium–adenosine triphosphatase (Na,K-ATPase) is a heterodimer of two subunits, α the catalytic subunit and the β subunit that stabilizes the protein and affects functional properties, together with a regulatory FXYD subunit (FXYD1–7) (2). There are four isoforms of α (α1−α4) and three isoforms of β (β1−β3). The α1β1 complex is the ubiquitous complex. α2 is expressed strongly in skeletal and cardiac muscle, brain astrocytes, and also ciliary epithelium. α3 is the neuronal form and α4 is restricted to testes (3). The ciliary epithelium is a syncitium consisting of an inner layer of pigmented epithelium (PE) facing the stroma and the outer nonpigmented epithelium (NPE) layer facing the aqueous humor. Na,K-ATPase is localized to the basolateral surface of both layers (4). Importantly, it is known that the primary isoform of Na,K-ATPase in PE is α1β1, whereas that in NPE is α2β3 in rodents (5).Cardiac glycosides (CGs) consist of a steroid core with a five- or six-membered unsaturated lactone ring and a variable number of sugars bound to the C3 position of the steroid. Each of these structural features has specific effects on the binding properties (6). Crystal structures of the Na,K-ATPase are available at moderate resolution in high-affinity complexes with ouabain (7) or bufalin and digoxin (8) and a low-affinity complex with bound K and ouabain (9). The high-affinity binding site is formed by a deep pocket comprised of transmembrane helices αTM1, 2, 4, 5, and 6, with the lactone-steroid moiety pointing inwards and sugars outwards. Binding of K⁺ occurs via backbone carbonyl oxygens in TM4 that also ligand the lactone moiety in the absence of K⁺, explaining the well-known antagonistic effect of K⁺ on CG binding (7–9).This paper focuses on α2β3-selective digoxin derivatives. The underlying hypothesis is that CGs with selectivity for the principal isoform complex in NPE cells, and sufficiently permeable to enter the eye when applied topically, could effectively inhibit aqueous humor inflow and reduce IOP. As background, digoxin shows moderate selectivity for the α2β1 isoform, and the selectivity is attributable to the tridigitoxose glycan moiety, as inferred from the fact that aglycones such as digoxigenin show no isoform selectivity (10). Following this work, we synthesized a series of perhydro-1,4-oxazepine derivatives of digoxin modified in the third sugar and showed that certain derivatives have enhanced selectivity for α2β1 (up to 7.5-fold) compared with digoxin itself (about fourfold) (11). The two most α2β1-selective derivatives effectively prevented or reversed a pharmacologically induced rise in IOP in rabbits.The current work is based on further insight regarding the β subunit, derived from a recent molecular structure of the Na,K-ATPase with bound digoxin (8). The glycan moiety of digoxin points outwards, and most interestingly, the third digitoxose approaches the β1 subunit near a single residue, Gln84 (Fig. 1). Gln84 in β1 is not conserved in β3 but is replaced by Val88 (Fig. 1). We hypothesized that diverse alkyl substitutions in the perhydro-1,4-oxazepine series of digoxin derivatives might interact with the alkyl side chain of Val88 and so raise selectivity for α2β3:α1β1. Several new alkyl derivatives do show strong selectivity for α2β3 over α1β1 and also potently reduce IOP in rabbits when applied topically.Open in a separate windowFig. 1.Model of human Na,K-ATPase α1β1 and α2β3 isoforms. The model depicts the human α1β1 complex and human α2β3 complex on the pig α1β1 structure with bound digoxin (4RET). α1 and α2 subunits, red; β1 subunit, gray; β3 subunit, blue; digoxin, green.     

Disruption of TAK1 in hepatocytes causes hepatic injury,inflammation, fibrosis,and carcinogenesis

TGF-β–activated kinase 1 (TAK1) is a MAP3K family member that activates NF-κB and JNK via Toll-like receptors and the receptors for IL-1, TNF-α, and TGF-β. Because the TAK1 downstream molecules NF-κB and JNK have opposite effects on cell death and carcinogenesis, the role of TAK1 in the liver is unpredictable. To address this issue, we generated hepatocyte-specific Tak1-deficient (Tak1ΔHEP) mice. The Tak1ΔHEP mice displayed spontaneous hepatocyte death, compensatory proliferation, inflammatory cell infiltration, and perisinusoidal fibrosis at age 1 month. Older Tak1ΔHEP mice developed multiple cancer nodules characterized by increased expression of fetal liver genes including α-fetoprotein. Cultures of primary hepatocytes deficient in Tak1 exhibited spontaneous cell death that was further increased in response to TNF-α. TNF-α increased caspase-3 activity but activated neither NF-κB nor JNK in Tak1-deficient hepatocytes. Genetic abrogation of TNF receptor type I (TNFRI) in Tak1ΔHEP mice reduced liver damage, inflammation, and fibrosis compared with unmodified Tak1ΔHEP mice. In conclusion, hepatocyte-specific deletion of TAK1 in mice resulted in spontaneous hepatocyte death, inflammation, fibrosis, and carcinogenesis that was partially mediated by TNFR signaling, indicating that TAK1 is an essential component for cellular homeostasis in the liver.     

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the α_vβ₃ integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with α_vβ₃ integrin or pretreatment of cells with a monoclonal antibody against α_vβ₃ integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.     

Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases. MUC5AC mucin is a major component of airway mucus, and its expression is modulated by a TNF-α-converting enzyme (TACE)–EGF receptor pathway that can be activated by reactive oxygen species (ROS). Dual oxidase 1 (Duox1), a homologue of glycoprotein p91^phox, is expressed in airway epithelium and generates ROS. We hypothesize that Duox1 activates TACE, cleaving pro-TGF-α into soluble TGF-α, resulting in mucin expression. To examine this hypothesis, we stimulated both normal human bronchial epithelial cells and NCI-H292 airway epithelial cells with phorbol 12-myristate 13-acetate and with human neutrophil elastase. These stimuli induced TACE activation, TGF-α release, and mucin expression, effects that were inhibited by ROS scavengers, implicating ROS in TACE activation. Inhibition of epithelial NADPH oxidase or knockdown of Duox1 expression with small interfering RNA prevented ROS generation, TGF-α release, and mucin expression by these stimuli, implicating Duox1 in TACE activation and mucin expression. Furthermore, the PKCδ/PKCθ inhibitor rottlerin prevented the effects induced by phorbol 12-myristate 13-acetate and human neutrophil elastase, suggesting that PKCδ and PKCθ are involved in Duox1 activation. From these results, we conclude that Duox1 plays a critical role in mucin expression by airway epithelial cells through PKCδ/PKCθ-Duox1-ROS-TACE-pro-ligand-EGF receptor cascade.     

RBBP9: A tumor-associated serine hydrolase activity required for pancreatic neoplasia

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-β-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-β signaling. Finally, RBBP9-mediated suppression of TGF-β signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell–cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.