Stabilization of integrin‐linked kinase by the Hsp90‐CHIP axis impacts cellular force generation,migration and the fibrotic response

Integrin‐linked kinase (ILK) is an adaptor protein required to establish and maintain the connection between integrins and the actin cytoskeleton. This linkage is essential for generating force between the extracellular matrix (ECM) and the cell during migration and matrix remodelling. The mechanisms by which ILK stability and turnover are regulated are unknown. Here we report that the E3 ligase CHIP–heat shock protein 90 (Hsp90) axis regulates ILK turnover in fibroblasts. The chaperone Hsp90 stabilizes ILK and facilitates the interaction of ILK with α‐parvin. When Hsp90 activity is blocked, ILK is ubiquitinated by CHIP and degraded by the proteasome, resulting in impaired fibroblast migration and a dramatic reduction in the fibrotic response to bleomycin in mice. Together, our results uncover how Hsp90 regulates ILK stability and identify a potential therapeutic strategy to alleviate fibrotic diseases.     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Over expression of integrin α 5 β 1 in human hepatocellular carcinoma cell line suppresses cell proliferation in vitro and tumorigenicity in nude mice

Integrin 5 1 and 2 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin 5 1 was decreased and another integrin 6 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin 5 and/or 1 cDNAs were established, and designated 5 1.6-7721, 5.3-7721, and 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin 5 and 1 subunits resulted in the overexpression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in 5.3-7721, 1.6-7721, 5 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with 1.6-7721,and 5 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin 5 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin 5 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.     

Differential requirements for ts-O45-G and procollagen biosynthetic transport

Emerging experimental evidence favours the existence of cargo sorting occurring upon the endoplasmic reticulum (ER) exit. Recent studies revealed that, in contrast to the conventional secretory marker ts-O45-G, procollagen (PC I) exits the ER at sites not coated with coat protein II and is transported to the Golgi complex in carriers devoid of coat protein I. Here, we investigated whether PC I trafficking requires a different molecular machinery in comparison with the ts-O45-G. By combining colocalization of the cargoes with endogenous markers, downregulation of transport machinery by RNA interference and knock-ins by complementary DNA over-expression, we provide strong evidence that PC I and ts-O45-G have common but also different molecular requirements during pre- and post-Golgi trafficking events.     

Proteotoxicity is not the reason for the dependence of cancer cells on the major chaperone Hsp70

Several years ago a hypothesis was proposed that the survival of cancer cells depend on elevated expression of molecular chaperones because these cells are prone to proteotoxic stress. A critical prediction of this hypothesis is that depletion of chaperones in cancer cells should lead to proteotoxicity. Here, using the major chaperone Hsp70 as example, we demonstrate that its depletion does not trigger proteotoxic stress, thus refuting the model. Accordingly, other functions of chaperones, e.g., their role in cell signaling, might define the requirements for chaperones in cancer cells, which is critical for rational targeting Hsp70 in cancer treatment.     

BIIB021, a novel Hsp90 inhibitor,sensitizes esophageal squamous cell carcinoma to radiation

BIIB021 is a novel, orally available inhibitor of heat shock protein 90 (Hsp90) that is currently in phase I/II clinical trials. BIIB021 induces the apoptosis of various types of tumor cells in vitro and in vivo. The aim of this study is to investigate the effect of BIIB021 on the radiosensitivity of esophageal squamous cell carcinoma (ESCC). The results indicated that BIIB021 exhibited strong antitumor activity in ESCC cell lines, either as a single agent or in combination with radiation. BIIB021 significantly downregulated radioresistant proteins including EGFR, Akt, Raf-1 of ESCC cell lines, increased apoptotic cells and enhanced G₂ arrest that is more radiosensitive cell cycle phase. These results suggest that this synthetic Hsp90 inhibitor simultaneously affects multiple pathways involved in tumor development and progression in the ESCC setting and may represent a better strategy for the treatment of ESCC patients, either as a monotherapy or a radiosensitizer.     

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

1. Download : Download high-res image (212KB)
2. Download : Download full-size image

     

The protein import machinery of the mitochondrial membranes

Mitochondria are surrounded by two membranes that contain independent and non-related protein transport machineries. Remarkable progress was recently achieved in elucidating the structure of the outer membrane import channel and in the identification of new components involved in protein traffic across the intermembrane space and the inner membrane. Traditional concepts of protein targeting and sorting had to be revised. Here we briefly summarize the data on the mitochondrial protein import system with particular emphasis on new developments and perspectives.     

Potential for targeting head and neck squamous cell carcinoma with monoclonal antibody K984

Summary In previous reports we have described the development of mAb K984, reactive with an epitope expressed on the outer cell surface of undifferentiated, proliferating cells in normal stratified squamous epithelia and their neoplastic counterparts [28, 30]. The K984 antigen was also found to be homogeneously expressed by in vitro cultured squamous cell carcinoma (SCC) cell lines. In the present study we demonstrate that mAb K984 induces a significant, dose-dependent growth inhibition when SCC cells are grown in vitro as monolayer cultures in the presence of mAb K984. These data seem to indicate that mAb K984 has potential for tumour targeting, especially in a therapeutic setting. As a first approach to evaluate the suitability of mAb K984 for tumour targeting in vivo, radiolabelled mAb K984 was administered to SCC-xenografted nude mice. Selective tumour accumulation of mAb K984 was observed. Tumour to blood ratios and tumour to non-tumour ratios, as based on the biodistribution data, were at least ten times higher in case of the specific mAb K984 when compared to another non-specific, isotypematched control antibody. mAb K984 was also capable of visualizing tumour deposits in xenografted nude mice. The corollary of these findings is that the mAb K984-defined antigen probably is involved in the regulation of proliferation of stratified squamous epithelium and squamous cell carcinoma and that mAb K984 has potential for specific tumour targeting.