Generation and identification of a conditional knockout allele for the PSMD11 gene in mice.

BACKGROUND: Our previous study have shown that the PSMD11 protein was an important survival factor for cancer cells except for its key role in regulation of assembly and activity of the 26S proteasome. To further investigate the role of PSMD11 in carcinogenesis, we constructed a conditional exon 5 floxed allele of PSMD11 (PSMD11flx) in mice. RESULTS: It was found that homozygous PSMD11 flx/flx mice showed normal and exhibited a normal life span and fertility, and showed roughly equivalent expression of PSMD11 in various tissues, suggesting that the floxed allele maintained the wild-type function. Cre recombinase could induce efficient knockout of the floxed PSMD11 allele both in vitro and in vivo. Mice with constitutive single allele deletion of PSMD11 derived from intercrossing between PSMD11flx/flx and CMV-Cre mice were all viable and fertile, and showed apparent growth retardation, suggesting that PSMD11 played a significant role in the development of mice pre- or postnatally. No whole-body PSMD11 deficient embryos (PSMD11-/-) were identified in E7.5-8.5 embryos in uteros, indicating that double allele knockout of PSMD11 leads to early embryonic lethality. To avoid embryonic lethality produced by whole-body PSMD11 deletion, we further developed conditional PSMD11 global knockout mice with genotype Flp;FSF-R26CAG - CreERT2/+; PSMD11 flx/flx, and demonstrated that PSMD11 could be depleted in a temporal and tissue-specific manner. Meanwhile, it was found that depletion of PSMD11 could induce massive apoptosis in MEFs. CONCLUSIONS: In summary, our data demonstrated that we have successfully generated a conditional knockout allele of PSMD11 in mice, and found that PSMD11 played a key role in early and postnatal development in mice, the PSMD11 flx/flx mice will be an invaluable tool to explore the functions of PSMD11 in development and diseases.

The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly.

BACKGROUND/AIMS: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patient-identified variants function molecularly in focal adhesions. METHODS: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. RESULTS: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. CONCLUSIONS: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients.

Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning.

Protein tagging with a wide variety of epitopes and/or fusion partners is used routinely to dissect protein function molecularly. Frequently, the required DNA subcloning is inefficient, especially in cases where multiple constructs are desired for a given protein with unique tags. Additionally, the generated clones have unwanted junction sequences introduced. To add versatile tags into the extracellular domain of the transmembrane protein THSD1, we developed a protein tagging technique that utilizes non-classical type IIS restriction enzymes that recognize non-palindromic DNA sequences and cleave outside of their recognition sites. Our results demonstrate that this method is highly efficient and can precisely fuse any tag into any position of a protein in a scarless manner. Moreover, this method is cost-efficient and adaptable because it uses commercially available type IIS restriction enzymes and is compatible with the traditional cloning system used by many labs. Therefore, precision tagging technology will benefit a number of researchers by providing an alternate method to integrate an array of tags into protein expression constructs.

Exercise training and return to a well-balanced diet activate the neuregulin 1/ErbB pathway in skeletal muscle of obese rats.

KEY POINTS: Some studies suggest that neuregulin 1 (NRG1) could be involved in the regulation of skeletal muscle energy metabolism in rodents. Here we assessed whether unbalanced diet is associated with alterations of the NRG1 signalling pathway and whether exercise and diet might restore NRG1 signalling in skeletal muscle of obese rats. We show that diet-induced obesity does not impair NRG1 signalling in rat skeletal muscle. We also report that endurance training and a well-balanced diet activate the NRG1 signalling in skeletal muscle of obese rats, possibly via a new mechanism mediated by the protease ADAM17. These results suggest that some beneficial effects of physical activity and diet in obese rats could be partly explained by stimulation of the NRG1 signalling pathway. ABSTRACT: Some studies suggest that the signalling pathway of neuregulin 1 (NRG1), a protein involved in the regulation of skeletal muscle metabolism, could be altered by nutritional and exercise interventions. We hypothesized that diet-induced obesity could lead to alterations of the NRG1 signalling pathway and that chronic exercise could improve NRG1 signalling in rat skeletal muscle. To test this hypothesis, male Wistar rats received a high fat/high sucrose (HF/HS) diet for 16 weeks. At the end of this period, NRG1 and ErbB expression/activity in skeletal muscle was assessed. The obese rats then continued the HF/HS diet or were switched to a well-balanced diet. Moreover, in both groups, half of the animals also performed low intensity treadmill exercise training. After another 8 weeks, NRG1 and ErbB expression/activity in skeletal muscle were tested again. The 16 week HF/HS diet induced obesity, but did not significantly affect the NRG1/ErbB signalling pathway in rat skeletal muscle. Conversely, after the switch to a well-balanced diet, NRG1 cleavage ratio and ErbB4 amount were increased. Chronic exercise training also promoted NRG1 cleavage, resulting in increased ErbB4 phosphorylation. This result was associated with increased protein expression and phosphorylation ratio of the metalloprotease ADAM17, which is involved in NRG1 shedding. Similarly, in vitro stretch-induced activation of ADAM17 in rat myoblasts induced NRG1 cleavage and ErbB4 activation. These results show that low intensity endurance training and well-balanced diet activate the NRG1-ErbB4 pathway, possibly via the metalloprotease ADAM17, in skeletal muscle of diet-induced obese rats.

TNFα shedding in mechanically stressed cardiomyocytes is mediated by Src activation of TACE.

Synthesized by the heart under hemodynamic overloading (mechanical stress), TNFα exerts complex effects on the heart -beneficial as a membrane protein and detrimental as a secreted protein, which presents a dilemma in the treatment of congestive heart failure. We postulate that by selectively blocking mechanical stress-induced cardiomyocyte secretion of TNFα, a function of TNFα converting enzyme (TACE), the detrimental effect of TNFα can be mitigated. However, the mechanism through which mechanical stress activates TACE in cardiomyocytes is unknown. Here, we report a molecular mechanism that mediates TACE activation in mechanically stressed cardiomyocytes. We found that the non-receptor tyrosine kinase Src mediates TACE activation in mechanically stretched rat cardiomyocytes by phosphorylating the Tyr-702 residue within the intracellular tail of TACE. The rapid activation of Src in mechanically stretched cardiomyocytes is followed by TACE phosphorylation on Tyr-702, leading to activation of p38 MAPK, a kinase that is an effector of TNFα receptor activation. Pharmacological inhibition or silencing of Src attenuated stretch-induced TACE phosphorylation on Tyr-702 and p38 activation. Overexpression of a TACE mutant in which Tyr-702 was replaced by alanine (TACE-Y702A) attenuated stretch-induced TNFα release from cardiomyocytes as well as activation of p38. These data suggests that Src mediates TACE activation in mechanically stressed cardiomyocytes and this mechanism could be exploited for specific blockade of TNFα secretion and its detrimental effects in congestive heart failure.

Src mediates the mechanical activation of myogenesis by activating TNFα-converting enzyme.

Mechanical stimulation affects many biological aspects in living cells through mechanotransduction. In myogenic precursor cells (MPCs), mechanical stimulation activates p38 mitogen-activated protein kinase (MAPK), a key regulator of myogenesis, via activating TNFα-converting enzyme (TACE, also known as ADAM17), to release autocrine TNFα. However, the signaling mechanism of mechanical activation of TACE is unknown. Because TACE possesses the structural features of substrates of the non-receptor tyrosine kinase Src, we tested the hypothesis that Src mediates mechanical activation of TACE in MPCs. We observed that mechanical stretch of C2C12 or primary rat myoblasts rapidly activates Src, which in turn interacts and colocalizes with TACE, resulting in tyrosine phosphorylation and activation of TACE. Particularly, Src activates TACE via the phosphorylation of amino acid residue Tyr702 in the intracellular tail of TACE, resulting in increased TNFα release and p38 activation. Src inhibition or deficiency blocks stretch activation of the TACE-p38-MAPK signaling, resulting in impaired myogenic gene expression. In response to functional overloading, Src and TACE are activated in mouse soleus muscle. Further, overloading-induced myogenesis and regeneration are impaired in the soleus of Src(+/-) mice. Therefore, Src mediates mechano-activation of TACE and myogenesis.

TIMP3: a physiological regulator of adult myogenesis.

Myogenic differentiation in adult muscle is normally suppressed and can be activated by myogenic cues in a subset of activated satellite cells. The switch mechanism that turns myogenesis on and off is not defined. In the present study, we demonstrate that tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of TNFalpha-converting enzyme (TACE), acts as an on-off switch for myogenic differentiation by regulating autocrine TNFalpha release. We observed that constitutively expressed TIMP3 is transiently downregulated in the satellite cells of regenerating mouse hindlimb muscles and differentiating C2C12 myoblasts. In C2C12 myoblasts, perturbing TIMP3 downregulation by overexpressing TIMP3 blocks TNFalpha release, p38 MAPK activation, myogenic gene expression and myotube formation. TNFalpha supplementation at a physiological concentration rescues myoblast differentiation. Similarly, in the regenerating soleus, overexpression of TIMP3 impairs release of TNFalpha and myogenic gene expression, and delays the formation of new fibers. In addition, downregulation of TIMP3 is mediated by the myogenesis-promoting microRNA miR-206. Thus, TIMP3 is a physiological regulator of myogenic differentiation.

Beta3-integrin mediates satellite cell differentiation in regenerating mouse muscle.

Skeletal muscle satellite cells can sense various forms of environmental cues and initiate coordinated signaling that activates myogenesis. Although this process involves cellular membrane receptor integrins, the role of integrins in myogenesis is not well defined. Here, we report a regulatory role of ß3-integrin, which was previously thought not expressed in muscle, in the initiation of satellite cell differentiation. Undetected in normal muscle, ß3-integrin expression in mouse hindlimb muscles is induced dramatically from 1 to 3 d after injury by cardiotoxin. The source of ß3-integrin expression is found to be activated satellite cells. Proliferating C2C12 myoblasts also express ß3-integrin, which is further up-regulated transiently on differentiation. Knockdown of ß3-integrin expression attenuates Rac1 activity, impairs myogenic gene expression, and disrupts focal adhesion formation and actin organization, resulting in impaired myoblast migration and myotube formation. Conversely, overexpression of constitutively active Rac1 rescues myotube formation. In addition, a ß3-integrin-neutralizing antibody similarly blocked myotube formation. Comparing with wild-type littermates, myogenic gene expression and muscle regeneration in cardiotoxin-injured ß3-integrin-null mice are impaired, as indicated by depressed expression of myogenic markers and morphological disparities. Thus, ß3-integrin is a mediator of satellite cell differentiation in regenerating muscle.

Diagnostic significance of serum type IV collagen (IVC) combined with aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio in liver fibrosis.

Background: Liver fibrosis is a necessary stage for various chronic liver diseases to develop into cirrhosis. The detection of serum markers of liver fibrosis is a commonly used method for early screening of liver fibrosis, mainly including type IV collagen (IVC), hyaluronic acid (HA), laminin (LN), and type III procollagen (PCIII). However, the high cost of the instrument and the slow detection speed are not conducive to mass screening and detection of the population. In this study, the widely used biochemical platform was used to jointly detect the liver fibrosis marker IVC and aspartate aminotransferase (AST) and alanine aminotransferase (ALT), We investigated the feasibility of serum IVC combined with the AST and ALT ratio (AST/ALT ratio) as a marker for liver fibrosis. Methods: A total of 81 patients with liver disease by clinical liver biopsy comprised the study group, and 50 healthy people who underwent physical examination in the study period were selected as the control group. Serum IVC, AST and ALT levels were detected by biochemical testing, AST/ALT ratio was calculated, and four serum markers of liver fibrosis (IVC, HA, LN, and PCIII) were measured by chemiluminescence. Moreover, imaging by color Doppler ultrasound (B-ultrasound) was performed for statistical analysis. Results: (I) Serum IVC and the AST/ALT ratio were significantly higher in the study group than in the control group (P<0.05). (II) The sensitivity of serum IVC combined with AST/ALT ratio detected biochemically and the four markers of liver fibrosis detected by chemiluminescence in the diagnosis of liver cirrhosis was 95.83%, 97.92%, without significant difference, but the specificity and accuracy of IVC + AST/ALT ratio in the diagnosis of liver cirrhosis were significantly higher (94.00%, 94.90%). (III) The detection rate of serum IVC + AST/ALT ratio for the diagnosis of early liver fibrosis was significantly higher than with imaging examination (45.45% vs. 21.21%). Conclusions: Serum IVC + AST/ALT ratio determined by biochemical analysis has high diagnostic accuracy in the diagnosis of liver fibrosis and liver cirrhosis, and is worthy of clinical application and promotion.

A hypersensitive estrogen receptor alpha mutation that alters dynamic protein interactions.

Estrogen receptor alpha (ERalpha) is highly regulated through multiple mechanisms including cell signaling, posttranslational modifications, and protein-protein interactions. We have previously identified a K303R ERalpha mutation within the hinge region of ERalpha. This mutation results in an altered posttranslational regulation and increased in vitro growth in the presence of low estrogen concentrations. We sought to determine if cells expressing this mutant ERalpha would display hypersensitive tumor growth in in vivo athymic ovariectomized nude mice. MCF-7 cells, stably expressing the K303R ERalpha, formed tumors in nude mice faster than cells expressing wild-type ERalpha in the presence of low levels of estrogen. When estrogen was withdrawn, all tumors regressed but half of the K303R ERalpha-expressing tumors became estrogen-independent and regrew. We evaluated potential mechanisms for the observed hypersensitivity. The mutant ERalpha did not demonstrate increased estrogen binding affinity, but did exhibit increased interactions with members of the SRC family of coactivators. The mutant ERalpha demonstrated increased levels and occupancy time on the pS2 promoter. In the presence of the K303R ERalpha, the SRC-3 and p300 coactivators also displayed increased levels and time on the pS2 promoter. The K303R ERalpha has, in part, lost critical negative regulation by the F domain. Collectively, these data demonstrate an important role for the K303R ERalpha mutation in hormonal regulation of tumor growth and estrogen-regulated promoter dynamics in human breast cancer.

Metastasis-associated protein 2 is a repressor of estrogen receptor alpha whose overexpression leads to estrogen-independent growth of human breast cancer cells.

Estrogen receptor (ER)alpha activity is controlled by the balance of coactivators and corepressors contained within cells that are recruited into transcriptional complexes. The metastasis-associated protein (MTA) family has been demonstrated to be associated with breast tumor cell progression and ERalpha activity. We demonstrate that MTA2 expression is correlated with ERalpha protein expression in invasive breast tumors. We show that the MTA2 family member can bind to ERalpha and repress its activity in human breast cancer cells. Furthermore, it can inhibit ERalpha-mediated colony formation and render breast cancer cells resistant to estradiol and the growth-inhibitory effects of the antiestrogen tamoxifen. MTA2 participates in the deacetylation of ERalpha protein, potentially through its associated histone deacetylase complex 1 activity. We hypothesize that MTA2 is a repressor of ERalpha activity and that it could represent a new therapeutic target of ERalpha action in human breast tumors.

Regulation of myogenic activation of p38 MAPK by TACE-mediated TNFα release.

The activation of p38 MAPK in myogenic precursor cells (MPCs) is a key signal for their exit of cell cycle and entry of the myogenic differentiation program. Therefore, identification of the signaling mechanism that activates p38 MAPK during this process is important for the understanding of the regulatory mechanism of muscle regeneration. This article reviews recent findings regarding the role of inflammatory cytokine tumor necrosis factor-α (TNFα) as a key activator of p38 MAPK during myogenesis in an autocrine/paracrine fashion, and the signaling mechanisms that converge upon TNFα converting enzyme (TACE) to release TNFα from differentiating MPCs in response to diverse regenerative stimuli.