Faqian Li

COP9 Signalosome Regulates Autophagosome Maturation

Background— Autophagy is essential to intracellular homeostasis and is involved in the pathophysiology of a variety of diseases. Mechanisms regulating selective autophagy remain poorly understood. The COP9 signalosome (CSN) is a conserved protein complex consisting of 8 subunits (CSN1 through CSN8), and is known to regulate the ubiquitin-proteasome system. However, it is unknown whether CSN plays a role in autophagy. Methods and Results— Marked increases in the LC3-II and p62 proteins were observed on Csn8 depletion in the cardiomyocytes of mouse hearts with cardiomyocyte-restricted knockout of the gene encoding CSN subunit 8 (CR-Csn8KO). The increases in autophagosomes were confirmed by probing with green fluorescent protein–LC3 and electron microscopy. Autophagic flux assessments revealed that defective autophagosome removal was the cause of autophagosome accumulation and occurred before a global ubiquitin-proteasome system impairment in Csn8-deficient hearts. Analyzing the prevalence of different stages of autophagic vacuoles revealed defective autophagosome maturation. Downregulation of Rab7 was found to colocalize strikingly with the autophagosome accumulation at the individual cardiomyocyte level. A significantly higher percent of cardiomyocytes with autophagosome accumulation underwent necrosis in CR-Csn8KO hearts. Long-term lysosomal inhibition with chloroquine induced cardiomyocyte necrosis in mice. Rab7 knockdown impaired autophagosome maturation of nonselective and selective autophagy and exacerbated cell death induced by proteasome inhibition in cultured cardiomyocytes. Conclusions— Csn8/CSN is a central regulator in not only the proteasomal proteolytic pathway, but also selective autophagy. Likely through regulating the expression of Rab7, Csn8/CSN plays a critical role in autophagosome maturation. Impaired autophagosome maturation causes cardiomyocytes to undergo necrosis.

Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung.

Differentiation of primary from metastatic adenocarcinoma in the lung can be challenging, and it demands sensitive and specific biomarkers, especially when the tissue for diagnosis is limited. Thyroid transcription factor-1 (TTF-1) has been considered a reliable marker for adenocarcinoma of lung origin. However, several recent studies have shown that TTF-1 immunostaining is also positive in adenocarcinomas arising in different organs including colon, endometrium, endocervix, and ovary. In addition, approximately 20% of lung primary adenocarcinomas are negative for TTF-1 immunostaining, and napsin A immunostaining has slightly higher sensitivity in detecting lung primary adenocarcinoma. We performed TTF-1 and napsin A immunostaining on 120 cases of primary lung adenocarcinomas and 37 cases of metastatic carcinomas in the lung. The results showed that 95 (79.2%) of 120 lung primary adenocarcinomas showed napsin A(+)/TTF-1(+) double-positive immunostaining pattern. TTF-1(−)/napsin A(+), TTF-1(+)/napsin A(−), and TTF-1(−)/napsin A(−) were seen in 8.3%, 3.3%, and 9.2% lung primary adenocarcinomas, respectively. Eight (21.6%) of the 37 metastatic carcinomas were positive for TTF-1 and they include clear-cell renal cell carcinomas completely negative for napsin A although napsin A was detected in 12 (80.0%) of 15 primary papillary and 3 (33.3%) of 9 primary clear-cell renal cell carcinomas. All renal epithelial neoplasms were TTF-1 negative. These findings indicate that double napsin A and TTF-1-positive immunostaining is highly specific for lung primary adenocarcinoma and the combination of these 2 biomarkers is warranted to help segregating primary lung adenocarcinoma from metastatic carcinoma in the lung.

A Crucial Role for p90RSK-Mediated Reduction of ERK5 Transcriptional Activity in Endothelial Dysfunction and Atherosclerosis

Background— Diabetes mellitus is a major risk factor for cardiovascular mortality by increasing endothelial cell (EC) dysfunction and subsequently accelerating atherosclerosis. Extracellular-signal regulated kinase 5 (ERK5) is activated by steady laminar flow and regulates EC function by increasing endothelial nitric oxide synthase expression and inhibiting EC inflammation. However, the role and regulatory mechanisms of ERK5 in EC dysfunction and atherosclerosis are poorly understood. Here, we report the critical role of the p90 ribosomal S6 kinase (p90RSK)/ERK5 complex in EC dysfunction in diabetes mellitus and atherosclerosis. Methods and Results— Inducible EC-specific ERK5 knockout (ERK5-EKO) mice showed increased leukocyte rolling and impaired vessel reactivity. To examine the role of endothelial ERK5 in atherosclerosis, we used inducible ERK5-EKO-LDLR−/− mice and observed increased plaque formation. When activated, p90RSK associated with ERK5, and this association inhibited ERK5 transcriptional activity and upregulated vascular cell adhesion molecule 1 expression. In addition, p90RSK directly phosphorylated ERK5 S496 and reduced endothelial nitric oxide synthase expression. p90RSK activity was increased in diabetic mouse vessels, and fluoromethyl ketone-methoxyethylamine, a specific p90RSK inhibitor, ameliorated EC-leukocyte recruitment and diminished vascular reactivity in diabetic mice. Interestingly, in ERK5-EKO mice, increased leukocyte rolling and impaired vessel reactivity were resistant to the beneficial effects of fluoromethyl ketone-methoxyethylamine, suggesting a critical role for endothelial ERK5 in mediating the salutary effects of fluoromethyl ketone-methoxyethylamine on endothelial dysfunction. Fluoromethyl ketone-methoxyethylamine also inhibited atherosclerosis formation in ApoE−/− mice. Conclusions— Our study highlights the importance of the p90RSK/ERK5 module as a critical mediator of EC dysfunction in diabetes mellitus and atherosclerosis formation, thus revealing a potential new target for therapeutic intervention.

Role of cAMP-Phosphodiesterase 1C Signaling in Regulating Growth Factor Receptor Stability, Vascular Smooth Muscle Cell Growth, Migration, and Neointimal Hyperplasia

RATIONALE Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). OBJECTIVE Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. METHODS AND RESULTS We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor β (PDGFRβ) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRβ, thus regulating PDGFRβ endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRβ degradation. CONCLUSIONS These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRβ protein degradation via low-density lipoprotein receptor-related protein-1.

Diagnostic utility of PAX8, TTF-1 and napsin A for discriminating metastatic carcinoma from primary adenocarcinoma of the lung.

Abstract TTF-1 and napsin A are useful biomarkers for differentiating primary lung adenocarcinoma from metastatic tumors. Studies have shown, however, that TTF-1 and napsin A also can be expressed in extrapulmonary carcinomas, and that a small fraction of primary lung adenocarcinomas do not co-express these two markers. We attempted to determine whether a tissue-specific transcriptional factor, PAX8, can help determine primary sites of lung carcinomas. Immunohistochemical stains for PAX8, TTF-1 and napsin A were performed on 103 cases of metastatic lung carcinomas from a variety of origins and 120 cases of primary lung adenocarcinomas. Our data demonstrated that all 103 metastatic carcinomas were negative for napsin A, while 14 (13.6%; four thyroid, two endometrium, three colon, one prostate, one salivary adenoid cystic, two renal cell carcinomas, and one ovary) showed weak to strong TTF-1 nuclear staining in 5–60% of the tumor cells. All primary lung adenocarcinomas were negative for PAX8, whereas 46 (44.7%) metastatic carcinomas from the kidney (29/33), ovary (6/8), endometrium (5/5), endocervix (1/1), thyroid (4/5) and urinary tract (1/3) were positive for PAX8. Our data demonstrate that of combined use of PAX8, TTF-1 and napsin A is reliable to separate reliably lung primary from metastatic tumors.

Reactive Oxygen Species Suppress Cardiac NaV1.5 Expression through Foxo1

NaV1.5 is a cardiac voltage-gated Na+ channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of NaV1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5′-CAAAACA-3′ (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5′-CAAAACAAAACA-3′. This finding led us to hypothesize that Foxo1 regulates NaV1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates NaV1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H2O2)-mediated NaV1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of NaV1.5 expression and Na+ current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of NaV1.5 expression. H2O2 significantly reduced NaV1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates NaV1.5 expression in cardiomyocytes and reactive oxygen species suppress NaV1.5 expression through Foxo1.

The COP9 Signalosome Is Required for Autophagy, Proteasome-Mediated Proteolysis, and Cardiomyocyte Survival in Adult Mice

Background—The COP9 signalosome (CSN) is an evolutionarily conserved protein complex composed of 8 unique protein subunits (CSN1 through CSN8). We have recently discovered in perinatal mouse hearts that CSN regulates not only proteasome-mediated proteolysis but also macroautophagy. However, the physiological significance of CSN in a post-mitotic organ of adult vertebrates has not been determined. We sought to study the physiological role of CSN8/CSN in adult mouse hearts. Methods and Results—Csn8 was conditionally ablated in the cardiomyocytes of adult mice (CSN8CKO) using a temporally controlled Cre-LoxP system. Loss of CSN8 accumulated the neddylated forms of cullins and noncullin proteins, increased ubiquitinated proteins, and stabilized a surrogate substrate of the proteasome in the heart. Autophagic flux was significantly decreased, whereas autophagosomes were markedly increased in CSN8CKO hearts, indicative of impaired autophagosome removal. Furthermore, we observed increased oxidized proteins, massive necrotic cardiomyocytes, and morphological and functional changes characteristic of dilated cardiomyopathy in CSN8CKO mice. Conclusions—CSN deneddylates substrates more than cullins and is indispensable to cardiomyocyte survival in not only perinatal hearts but also adult hearts. CSN8/CSN regulates both proteasome-mediated proteolysis and the autophagic-lysosomal pathway, critical to the removal of oxidized proteins in the heart.

Canonical Wnt/β-catenin signaling in epicardial fibrosis of failed pediatric heart allografts with diastolic dysfunction.

BACKGROUND Failed pediatric heart allografts with diastolic dysfunction exhibit severe epicardial fibrosis. The molecular mechanism underlying this process is poorly understood. Canonical Wnt/β-catenin signaling plays an important role in epithelial-mesenchymal transition and is implicated in fibrosing diseases. In this study, we tested the hypothesis that canonical Wnt/β-catenin signaling is activated in epicardial fibrosis of end-stage dysfunctional pediatric allografts. METHODS Fourteen explanted heart grafts of 12 patients who had undergone 14 heart transplantations were used for immunohistochemical staining of β-catenin and its nuclear binding partners, T-cell factor/lymphoid enhancer factor family transcriptional factors. Fourteen age-matched native hearts from patients who had undergone first heart transplantation without evidence of epicardial fibrosis were used as controls. RESULTS AND CONCLUSIONS Epicardial fibroblasts from explanted allografts demonstrated nuclear accumulation of β-catenin. These cells also showed nuclear positivity for T-cell factor 4. No T-cell factor 3 expression was present in the epicardium. T-cell factor 1 and lymphoid enhancer factor 1 were observed in lymphocytes, but not in other cell types of the epicardium. These findings suggest an association between canonical Wnt/beta-catenin signaling and epicardial fibrosis of failed pediatric heart allografts. Should activation of this pathway be shown to be causal to epicardial fibrosis in this setting, then inhibition of this pathway may help to prevent this devastating process.

High-grade neuroendocrine carcinomas of the lung highly express enhancer of zeste homolog 2, but carcinoids do not

Enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, is a histone methyltransferase and plays an important role in cell proliferation and cell cycle regulation. It has been shown to be overexpressed in a number of malignant neoplasms. This study aimed to determine the expression pattern of enhancer of zeste homolog 2 in neuroendocrine tumors of the lung and the potential of enhancer of zeste homolog 2 to serve as a biomarker to segregate carcinoids from high-grade neuroendocrine carcinomas. Fifty-four cases, including 25 typical carcinoids, 7 atypical carcinoids, 9 large-cell neuroendocrine carcinomas, and 13 small-cell lung carcinomas, were immunohistochemically studied using a monoclonal antibody against enhancer of zeste homolog 2. All 13 small-cell lung carcinomas demonstrated moderate to strong nuclear staining with 12 exhibiting more than 90% of tumor cells staining. All 9 large-cell neuroendocrine carcinomas were moderately to strongly positive for enhancer of zeste homolog 2, with 6 cases having staining in more than 80% of tumor cells. In contrast, all 25 typical carcinoids and 6 atypical carcinoids showed only rare scattered enhancer of zeste homolog 2-positive tumor cells, with 1 case of atypical carcinoid exhibiting moderate staining in 40% of tumor cells. A subsequent validation study of the 14 specimens of lung or mediastinal lymph node biopsy and fine-needle aspiration, including 6 small-cell lung carcinomas, 2 large-cell neuroendocrine carcinomas, 5 typical carcinoids, and 1 atypical carcinoid, was performed. Enhancer of zeste homolog 2 was diffusely and strongly positive in all small-cell lung carcinomas and large-cell neuroendocrine carcinomas, even with severe crush artifact, whereas it was only positive in rare tumor cells in carcinoids. These findings support the formulation that enhancer of zeste homolog 2 may play an important role in the regulation of biologic behavior of high-grade neuroendocrine carcinomas and as a diagnostically useful marker in distinguishing high-grade neuroendocrine carcinomas from carcinoids.

COP9 signalosome subunit 8 is required for postnatal hepatocyte survival and effective proliferation

Studies using lower organisms and cultured mammalian cells have revealed that the COP9 signalosome (CSN) has important roles in multiple cellular processes. Conditional gene targeting was recently used to study CSN function in murine T-cell development and activation. Using the Cre-loxP system, here we have achieved postnatal hepatocyte-restricted knockout of the csn8 gene (HR-Csn8KO) in mice. The protein abundance of other seven CSN subunits was differentially downregulated by HR-Csn8KO and the deneddylation of all cullins examined was significantly impaired. Moreover, HR-Csn8KO-induced massive hepatocyte apoptosis and evoked extensive reparative responses in the liver, including marked intralobular proliferation of biliary lineage cells and trans-differentiation and proliferation of the oval cells. However, division of pre-existing hepatocytes was significantly diminished in HR-Csn8KO livers. These findings indicate that Csn8 is essential to the ability of mature hepatocytes to proliferate effectively in response to hepatic injury. The histopathological examinations revealed striking hepatocytomegaly in Csn8-deficient livers. The hepatocyte nuclei were dramatically enlarged and pleomorphic with hyperchromasia and prominent nucleoli, consistent with dysplasia or preneoplastic cellular alteration in HR-Csn8KO mice at 6 weeks. Pericellular and perisinusoid fibrosis with distorted architecture was also evident at 6 weeks. It is concluded that CSN8/CSN is essential to postnatal hepatocyte survival and effective proliferation.