Alveolar type 2 (AT2) cell dysfunction is key to the development of many lung diseases including pulmonary fibrosis (PF). The underlying mechanisms often remain poorly understood due to a paucity of manipulable primary human models and lack of defined triggers. Monogenic forms of familial pulmonary fibrosis combined with recent advances in in vitro culture techniques offer a unique opportunity to interrogate early pathogenic events in PF. To model toxic gain-of-function disease caused by the SFTPC variant I73T, we employed a base editing strategy in human lung-derived AT2 (fdAT2) organoids to edit the endogenous SFTPC locus and generate a heterozygous SFTPC-I73T-expressing disease model which we interrogated when grown in standard 3D culture, monolayer culture, and at air-liquid interface. SFTPC-I73T expressing organoids failed to form lumens and displayed disrupted epithelial polarity. This was due to SFTPC accumulation in enlarged early endosomes resulting in impaired apico-basal trafficking of polarity and adhesion proteins. Air exposure exacerbated these defects, causing epithelial barrier breakdown and impaired wound healing. Together, we demonstrate the ability to edit endogenous loci in differentiated alveolar organoids to generate disease models that provide mechanistic insights into disease. We establish endosomal dysfunction and polarity loss as drivers of SFTPC-I73T-mediated epithelial injury and highlight mechanisms that may underlie AT2 dysfunction in disease more broadly.

Impaired proteostasis is a cellular hallmark of aging, and the ubiquitinproteasome system is a fundamental driver of proteostasis. As an E3 ubiquitin ligase, WW-domain containing protein 1 (WWP1) expression and activity are tightly regulated in cells, while its deregulation has been described in cancer, in neurodegenerative diseases, and in heart failure. However, the protein-protein interaction network of WWP1 is understudied, particularly in the heart. Here, we conducted a yeast-two hybrid (Y2H) screen of a human heart library and identified 21 putative WWP1 interactors, including 12 whose expression and potential function in the heart were previously unappreciated. Central in the identified protein-protein interaction network was WBP2 (WW domain binding protein 2), an oncogenic transcriptional co-activator. Utilizing immunofluorescence and proximity ligation assays, it was confirmed that endogenous WWP1 can co-localize and interact with WBP2 in human heart tissue, and, using the Y2H system, we showed that this interaction is dependent upon the associations between WW domains 1 and 3 from WWP1 and PY domains 2 and 3 of WBP2. In total, these data serve as a launching pad to identify broader protein networks regulated by WWP1 and the regions of interaction which might be targetable to reduce hallmarks of cellular aging.

Alveolar type 2 (AT2) cells maintain lung health by acting as stem cells and producing pulmonary surfactant. AT2 dysfunction underlies many lung diseases, including interstitial lung disease (ILD), in which some inherited forms result from the mislocalization of surfactant protein C (SFTPC) variants. Lung disease modeling and dissection of the underlying mechanisms remain challenging due to complexities in deriving and maintaining human AT2 cells ex vivo. Here, we describe the development of mature, expandable AT2 organoids derived from human fetal lungs which are phenotypically stable, can differentiate into AT1-like cells, and are genetically manipulable. We use these organoids to test key effectors of SFTPC maturation identified in a forward genetic screen including the E3 ligase ITCH, demonstrating that their depletion phenocopies the pathological SFTPC redistribution seen for the SFTPC-I73T variant. In summary, we demonstrate the development of a novel alveolar organoid model and use it to identify effectors of SFTPC maturation necessary for AT2 health.

Background:Idiopathic pulmonary fibrosis is a fatal lung disease of progressive lung parenchymal scarring caused by the aberrant response of an alveolar epithelium repeatedly exposed to injury. Understanding epithelial dysfunction has been hampered by the lack of physiological alveolar type 2 (AT2) cell models and defined disease triggers. Monogenic forms of familial pulmonary fibrosis (FPF) caused by toxic gain-of-function variants provide an opportunity to investigate early pathogenic events. One such variant, surfactant protein C (SFTPC)-I73T, abnormally localises within AT2 cells and causes their dysfunction. Methods:We used base editing of fetal lung-derived AT2 (fdAT2) organoids to create a heterozygous disease model of endogenous SFTPC-I73T expression. We also created an inducible overexpression system to interrogate temporal changes associated with SFTPC-I73T expression. We cultured fdAT2 both in 3D culture and at air-liquid interface to understand the importance of polarity cues and air exposure on disease phenotypes. Results:In our heterozygous endogenous expression system, we found that fdAT2 expressing SFTPC-I73T grew without a lumen and were unable to correctly polarise. SFTPC-I73T accumulated with time and caused gross enlargement of early endosomes, preventing correct apico-basal trafficking of multiple endosomally trafficked cargoes including polarity markers and cell adhesion proteins. This phenotype was exacerbated by air exposure and led to loss of epithelial monolayer integrity and abnormal wound healing after injury. Conclusion:Using endogenous gene editing for the first time in differentiated alveolar organoids, we have demonstrated that the pathogenic effects of SFTPC-I73T are mediated through endosomal dysfunction and abnormal epithelial organisation. This has important implications for AT2 function in vivo.

Supplementary Figure 2 from American Ginseng Suppresses Colitis through p53-Mediated Apoptosis of Inflammatory Cells

Supplementary Figure 1 from American Ginseng Suppresses Colitis through p53-Mediated Apoptosis of Inflammatory Cells

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pervasive event in tumorigenesis due to PI3K mutation and dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Pharmacological inhibition of PI3K has resulted in variable clinical outcomes, however, raising questions regarding the possible mechanisms of unresponsiveness and resistance to treatment. WWP1 is an oncogenic HECT-type ubiquitin E3 ligase frequently amplified and mutated in multiple cancers, as well as in the germ lines of patients predisposed to cancer, and was recently found to activate PI3K signaling through PTEN inactivation. Here, we demonstrate that PTEN dissociated from the plasma membrane upon treatment with PI3K inhibitors through WWP1 activation, whereas WWP1 genetic or pharmacological inhibition restored PTEN membrane localization, synergizing with PI3K inhibitors to suppress tumor growth both in vitro and in vivo. Furthermore, we demonstrate that WWP1 inhibition attenuated hyperglycemia and the consequent insulin feedback, which is a major tumor-promoting side effect of PI3K inhibitors. Mechanistically, we found that AMPKα2 was ubiquitinated and, in turn, inhibited in its activatory phosphorylation by WWP1, whereas WWP1 inhibition facilitated AMPKα2 activity in the muscle to compensate for the reduction in glucose uptake observed upon PI3K inhibition. Thus, our identification of the cell-autonomous and systemic roles of WWP1 inhibition expands the therapeutic potential of PI3K inhibitors and reveals new avenues of combination cancer therapy.

Background. Heart failure with a preserved left ventricular (LV) ejection fraction (HFpEF) often arises from a prolonged LV pressure overload (LVPO) and accompanied by abnormal extracellular matrix (ECM) accumulation. The E3 ubiquitin ligase WWP1 is a fundamental determinant ECM turnover. We tested the hypothesis that genetic ablation of Wwp1 would alter the progression of LVPO induced HFpEF. Methods/Results. LV echocardiography in mice with global Wwp1 deletion (n=41; Wwp1-/-) was performed at 12 weeks of age (Baseline) and then at 2 and 4 weeks following LVPO (transverse aortic banding) or surgery without LVPO induction. Age-matched wild type mice (Wwp1+/+; n=33) underwent identical protocols. LV EF remained constant and unchanged with LVPO and LV mass increased in both groups but was lower in the Wwp1-/- mice. With LVPO, the E/A ratio, an index of LV filling, was 3.97 + 0.46 in Wwp1+/+ but was 1.73 + 0.19 in the Wwp1-/- group (p<0.05). At the transcriptional level, mRNA for fibrillar collagens (types I and III) decreased by approximately 50% in Wwp1-/- compared to the Wwp1+/+ group at 4 weeks post-LVPO (p<0.05) and was paralleled by a similar difference in LV fibrillar collagen content as measured by histochemistry. Moreover, mRNA levels for determinants favoring ECM accumulation, such as transforming growth factor (TGF) increased with LVPO, but were lower in the Wwp1-/- group. Summary. The absence of Wwp1 reduced the development of LVH and subsequent progression to HFpEF. Modulating the WWP1 pathway could be a therapeutic target to alter the natural history of HFpEF.

Aims. Ubiquitylation is a key event that regulates protein turnover, and induction of the ubiquitin ligase E3 WWP1 has been associated with age. Left ventricular hypertrophy (LVH) commonly occurs as a function of age and can cause heart failure with a preserved ejection fraction (EF; HFpEF). We hypothesized that overexpression (O/E) of WWP1 in the heart would cause LVH as well as functional and structural changes consistent with the aging HFpEF phenotype. Methods and Results. Global WWP1 O/E was achieved in mice (n=11) and echocardiography (40 MHz) performed to measure LV mass, EF, Doppler velocities (early-E, late/atrial-A), myocardial relaxation (E’), and isovolumetric relaxation time (IVRT) at 4, 6, and 8 weeks. Age matched wild type animals (n=15) were included as referent controls. LV EF was identical (60+1% vs 60+1%, p>0.90) with no difference in LV mass (67+3 vs 75+5, p>0.25) at 4 weeks. However, at 8 weeks of age, LV mass increased by over two-fold, E/A fell (impaired passive filling), and E/E’ was lower and IVRT prolonged (impaired LV relaxation) - all p<0.05. Collagen percent area increased by over two-fold and fibrillar collagen expression (rtPCR) by over 1.5 fold (p<0.05) with WWP1 O/E. WWP1 with an anti-WWP1 antibody could be identified in isolated cardiac fibroblasts with WWP1 increased by over two-fold in O/E fibroblasts (p<0.05). Conclusion. Inducing WWP1 expression caused LVH, preserved systolic function, but impaired diastolic dysfunction, consistent with the HFpEF phenotype. Targeting the WWP1 pathway may be a novel therapeutic target for this intractable form of HF associated with aging.

Heart failure (HF) with a preserved ejection fraction (HFpEF) is a growing cause of HF and commonly afflicts the elderly. Milestones for HFpEF include diastolic dysfunction and an abnormal extracellular matrix (ECM). The ubiquitin ligases, such as WWP1, change with aging and regulate critical protein turnover/stability processes, such as the ECM. The present study demonstrated that induction of WWP1 in mice induced LV hypertrophy, diastolic dysfunction, and ECM accumulation, consistent with the HFpEF phenotype, and thus may identify a new therapeutic pathway.

Activation of tumor suppressors for the treatment of human cancer has been a long sought, yet elusive, strategy. PTEN is a critical tumor suppressive phosphatase that is active in its dimer configuration at the plasma membrane. Polyubiquitination by the ubiquitin E3 ligase WWP1 (WW domain-containing ubiquitin E3 ligase 1) suppressed the dimerization, membrane recruitment, and function of PTEN. Either genetic ablation or pharmacological inhibition of WWP1 triggered PTEN reactivation and unleashed tumor suppressive activity. WWP1 appears to be a direct MYC (MYC proto-oncogene) target gene and was critical for MYC-driven tumorigenesis. We identified indole-3-carbinol, a compound found in cruciferous vegetables, as a natural and potent WWP1 inhibitor. Thus, our findings unravel a potential therapeutic strategy for cancer prevention and treatment through PTEN reactivation.

Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha(18H/a18H)). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67(+) proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha(18H/a18H) animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha(18H/a18H) mice carrying the Min mutation (Itcha(18H/a18H); Apc(Min) (/+)) displayed a 76% reduction in tumor burden as compared to Apc(Min) (/+) littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.

Introduction: Cardiomyocyte hypertrophy is observed during normal postnatal cardiac development as well as during disease-related cardiac remodeling. This growth is accompanied by dynamic changes at the intercalated disc, including the redistribution and degradation of the gap junction protein connexin 43 (Cx43). We have shown that the ubiquitin ligase Wwp1 can promote the degradation of Cx43, and that genetic upregulation of Wwp1 stimulates hypertrophy and arrhythmogenesis. Hypothesis: We hypothesized that changes in the expression of Wwp1 would be associated with developmental and pathological hypertrophy programs. Methods: Expression of Wwp1 was analyzed by digital droplet PCR in hearts derived from normally developing mice at 2, 4, 6, and 8 weeks of age (n=6 for each) or from adult mice subjected to transverse aortic constriction (n=5) vs. sham operated animals (n=5). Cardiac structure and function of mice overexpressing or lacking expression of Wwp1 was evaluated by transmission electron microscopy (n=3) and echocardiography (n=6). Results: The expression of Wwp1 during postnatal development was very dynamic, peaking at a concentration of 324 copies/uL in the hearts derived from 4 week old animals. This also corresponded to the timepoint when the gap junction length at the intercalated disc plateaued. Further, a 1.5-fold increase in the expression of Wwp1 in animals subjected to pressure overload was detected as compared to animals subjected to surgery without pressure overload (p<0.007). Interestingly, the concentration of Wwp1 message in pressure overloaded animals (331 copies/uL) was nearly identical to that found in 4 week old wild type animals during normal postnatal development. Consistent with the level of Wwp1 correlating with cardiomyocyte hypertrophy, animals lacking Wwp1 had a 23-42% decrease in left ventricular mass (p<0.01) whereas transgenic animals overexpressing Wwp1 had a 28-50% increase in left ventricular mass (p<0.005) as compared to wild type controls between 4 and 8 weeks of age. Conclusions: This study shows that expression of Wwp1 is upregulated during hypertrophy and this correlates with ultrastructural changes at the intercalated disc.

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a β-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and –independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.

Major progress in deciphering the role of the E3 ligase, ITCH, in animal physiology has come from the generation and identification of Itch loss-of-function mutant mice (itchy). Mutant mice display an autoimmune-like phenotype characterized by chronic dermatitis, which has been attributed to increased levels of ITCH target proteins (e.g. transcription factors JUNB and CJUN) in T cells. Autoimmune disorders also exist in humans with Itch frameshift mutations resulting in loss of functional ITCH protein. Recent phenotypic analysis of male itchy mice revealed reduced sperm production, although cross breeding experiments showed no difference in litter size when male itchy mice were bred to wild type females. However, a reduction in litter sizes did occur when itchy females were bred to wild type males. Based on these results, characterization of female reproductive function in itchy mice was performed. Developmental analysis of fetuses at gestational day 18.5, cytological evaluation of estrous cyclicity, histopathological analysis of ovaries, and protein analysis were used to investigate the itchy reproductive phenotype. Gross skeletal and soft tissue analysis of gestational day 18.5 itchy fetuses indicated no gross developmental deformities. Itchy females had reduced implantation sites, decreased corpora lutea, and increased estrous cycle length due to increased number of days in estrus compared to controls. Alterations in the expression of prototypical ITCH targets in the ovaries were not indicated, suggesting that an alteration in an as yet defined ovary-specific ITCH substrate or interaction with the altered immune system likely accounts for the disruption of female reproduction. This report indicates the importance of the E3 ligase, ITCH, in female reproduction.

Itch is a ubiquitin E3 ligase that regulates protein stability. Itch(-/-) mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch(-/-) bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-kappa B ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcription factors c-fos and Nfatc1. Overexpression of Itch in Itch(-/-) cells rescued increased osteoclastogenesis. RANKL increased Itch expression, which can be blocked by a NF-kappa B inhibitor. The murine Itch promoter contains NF-kappa B binding sites. Overexpression of NF-kappa B p65 increased Itch expression, and RANKL promoted the binding of p65 onto the NF-kappa B binding sites in the Itch promoter. Itch(-/-) osteoclast precursors had prolonged RANKL-induced NF-kappa B activation and delayed TNF receptor-associated factor 6 (TRAF6) deubiquitination. In WT osteoclast precursors, Itch bound to TRAF6 and the deubiquitinating enzyme cylindromatosis. Adult Itch(-/-) mice had normal bone volume, but they had significantly increased LPS-induced osteoclastogenesis and bone resorption. Thus, Itch is a new RANKL target gene that is induced during osteoclastogenesis. Itch interacts with the deubiquitinating enzyme and is required for deubiquitination of TRAF6, thus limiting RANKL-induced osteoclast formation.

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

Abstract Somatic mutations resulting in constitutive activation of the CARMA1 scaffolding protein occur frequently in poor prognosis diffuse large B cell lymphoma (DLBCL). Such mutations directly promote assembly of the CARMA1, BCL10, MALT1 (CBM complex) thereby leading to activation of the IKK signalosome. These events lead to sustained NF-κB signaling, which is essential for lymphocyte survival. Thus, understanding the events that down-regulate CBM complex assembly is predicted to identify novel therapeutic targets in DLBCL. Notably, stimulation of antigen receptors simultaneously triggers both CARMA1-dependent BCL10 activation and CARMA-dependent-BCL10 degradation, which ultimately limits CARMA1 signaling. Using mutant B cell models, and genetic as well as biochemical approaches, we identify a kinase-independent requirement for the kinase, TAK1, and its adaptor, TAB1, in antigen receptor-induced BCL10 proteolysis. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 over-expression restrains CARMA1-dependent NF-κB activation by reducing BCL10 levels. TAK1 also promotes counter-selection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counter-balances NF-κB and JNK signals essential for activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

Rationale: The electrical coupling between adjacent cardiomyocytes is tightly regulated by gap junctions (GJ) to ensure proper electrical conduction in the myocardium. We have recently shown that the ubiquitin ligase Wwp1 can interact with and ubiquitylate the GJ protein connexin 43 (Cx43) causing its degradation. All mice ubiquitously overexpressing Wwp1 develop left ventricular hypertrophy (LVH), a 90% decrease in Cx43 protein, and die from sudden cardiac death (SCD) around 8 weeks of age. Hypothesis: We hypothesize that the effects of Wwp1 overexpression are cell autonomous to cardiomyocytes and that some degree of compensation exists until 8 weeks of age in mice. Methods: Cardiomyocyte-specific overexpression of Wwp1 was achieved using a tamoxifen-inducible cre transgenic mouse line driven by the α-MHC promoter. LVH was assessed in animals overexpressing cardiac Wwp1 (n=8) relative to their wild type littermates (n=8) using echocardiography. Cx43 and Cx45 protein levels and localization within myocardial tissue was determined via Western blotting and multi-immunofluorescence staining in conjunction with laser scanning confocal microscopy. Susceptibility to arrhythmias was ascertained using ventricular burst pacing monitored by ECG. Results: Induction of cardiomyocyte-specific Wwp1overexpression at 4 weeks of age resulted in decreased Cx43 expression, ECG abnormalities, and inducible ventricular arrhythmias as early as 48h following induction. In contrast, protein levels of Cx45 were unaltered but showed mislocalization. Unpaced Wwp1 overexpressers (n=12) displayed progressive LVH and SCD at 8 weeks of age. Interestingly, induction of cardiac Wwp1 overexpression at 8 weeks of age or later resulted in similar Cx43 and Cx45 changes and associated ECG abnormalities. Spontaneous or induced ventricular arrhythmias were observed as early as 48h following induction with no associated LVH. Conclusions: Wwp1 exerts its effects on cardiac structure and function in a cell autonomous manner. There is some plasticity in the myocardium which provides protection against spontaneous arrhythmias up until 8 weeks of age in mice. This mechanistic insight highlights the potential for novel anti-arrhythmia therapeutics targeting Wwp1.