NgBR is essential for endothelial cell glycosylation and vascular development

NgBR is a transmembrane protein identified as a Nogo‐B‐interacting protein and recently has been shown to be a subunit required for cis‐prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial‐specific NgBR knockout embryos. Here, we show that endothelial‐specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE‐cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo‐B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.     

The Nogo‐B receptor promotes human hepatocellular carcinoma cell growth via the Akt signal pathway

Nogo‐B receptor (NgBR) is a type I receptor with a single transmembrane domain and specifically binds to ligand Nogo‐B. A previous study demonstrated that NgBR was highly expressed in human breast invasive ductal carcinoma and promoted epithelial‐mesenchymal transition in breast tumor cells. Our recent work found that NgBR expression was associated with a poor prognosis in human patients with hepatocellular carcinoma (HCC). Here, we elucidate that the increased expression of NgBR contributes toward the increased cell growth of human HCC cells both in vitro and in vivo. Cell viability and clonogenic survival analysis results demonstrated that knockdown of NgBR inhibits the cell growth in human HCC cells, which correlates with a reduction in the phosphorylation of Akt levels. Furthermore, overexpression of NgBR by the cotransfected pIRES‐NgBR plasmid together with NgBR siRNA in human HCC cells can rescue impaired phosphorylation of Akt levels in NgBR knockdown human HCC cells. In addition, cell viability analyses showed that NgBR overexpression can rescue the cell growth inhibition presented in human HCC NgBR knockdown cells. Taken together, our results suggest that NgBR potentially acts as an oncogene in HCC by increasing Akt activity. Thus, NgBR may represent a new potential diagnostic and therapeutic target for the treatment of HCC.     

Activation of hepatic Nogo-B receptor expression—A new anti-liver steatosis mechanism of statins

Deficiency of hepatic Nogo-B receptor (NgBR) expression activates liver X receptor α (LXRα) in an adenosine monophosphate-activated protein kinase α (AMPKα)-dependent manner, thereby inducing severe hepatic lipid accumulation and hypertriglyceridemia. Statins have been demonstrated non-cholesterol lowering effects including anti-nonalcoholic fatty liver disease (NAFLD). Herein, we investigated if the anti-NAFLD function of statins depends on activation of NgBR expression. In vivo, atorvastatin protected apoE deficient or NgBR floxed, but not hepatic NgBR deficient mice, against Western diet (WD)-increased triglyceride levels in liver and serum. In vitro, statins reduced lipid accumulation in nonsilencing small hairpin RNA-transfected (shNSi), but not in NgBR small hairpin RNA-transfected (shNgBRi) HepG2 cells. Inhibition of cellular lipid accumulation by atorvastatin is related to activation of AMPKα, and inactivation of LXRα and lipogenic genes. Statin also inhibited expression of oxysterol producing enzymes. Associated with changes of hepatic lipid levels by WD or atorvastatin, NgBR expression was inversely regulated. At cellular levels, statins increased NgBR mRNA and protein expression, and NgBR protein stability. In contrast to reduced cellular cholesterol levels by statin or β-cyclodextrin, increased cellular cholesterol levels decreased NgBR expression suggesting cholesterol or its synthesis intermediates inhibit NgBR expression. Indeed, mevalonate, geranylgeraniol or geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate or farnesol, blocked atorvastatin-induced NgBR expression. Furthermore, we determined that induction of hepatic NgBR expression by atorvastatin mainly depended on inactivation of extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (Akt). Taken together, our study demonstrates that statins inhibit NAFLD mainly through activation of NgBR expression.     

Nogo-B receptor possesses an intrinsically unstructured ectodomain and a partially folded cytoplasmic domain

RTN4/Nogo proteins containing three isoforms have been implicated in a large and diverse spectrum of biological functions. By contrast, only two functional receptors were known for them, namely NgR binding the 66-residue ectodomain shared by all three Nogos and NgBR specifically binding Nogo-B. The 297-residue NgBR was recently identified to be essential for stimulating chemotaxis and morphogenesis of endothelial cells but its structural property still remains completely unknown. In the present study, we expressed and subsequently conducted bioinformatics, CD and NMR characterization of NgBR and its two dissected domains. Very surprisingly, our results indicate that the NgBR ectodomain is intrinsically unstructured without both secondary and tertiary structures while the cytoplasmic domain is only partially folded with secondary structures but without a tight tertiary packing. Therefore, NgBR is a very rare example showing that the entire ectodomain of a transmembrane receptor could be predominantly disordered and the results presented here may bear important implications in understanding NgBR functions in the future.     

Expression of NgBR Is Highly Associated with Estrogen Receptor Alpha and Survivin in Breast Cancer

NgBR is a type I receptor with a single transmembrane domain and was identified as a specific receptor for Nogo-B. Our recent findings demonstrated that NgBR binds farnesylated Ras and recruits Ras to the plasma membrane, which is a critical step required for the activation of Ras signaling in human breast cancer cells and tumorigenesis. Here, we first use immunohistochemistry and real-time PCR approaches to examine the expression patterns of Nogo-B and NgBR in both normal and breast tumor tissues. Then, we examine the relationship between NgBR expression and molecular subtypes of breast cancer, and the roles of NgBR in estrogen-dependent survivin signaling pathway. Results showed that NgBR and Nogo-B protein were detected in both normal and breast tumor tissues. However, the expression of Nogo-B and NgBR in breast tumor tissue was much stronger than in normal breast tissue. The statistical analysis demonstrated that NgBR is highly associated with ER-positive/HER2-negative breast cancer. We also found that the expression of NgBR has a strong correlation with the expression of survivin, which is a well-known apoptosis inhibitor. The correlation between NgBR and survivin gene expression was further confirmed by real-time PCR. In vitro results also demonstrated that estradiol induces the expression of survivin in ER-positive T47D breast tumor cells but not in ER-negative MDA-MB-468 breast tumor cells. NgBR knockdown with siRNA abolishes estradiol-induced survivin expression in ER-positive T47D cells but not in ER-negative MDA-MB-468 cells. In addition, estradiol increases the expression of survivin and cell growth in ER-positive MCF-7 and T47D cells whereas knockdown of NgBR with siRNA reduces estradiol-induced survivin expression and cell growth. In summary, these results indicate that NgBR is a new molecular marker for breast cancer. The data suggest that the expression of NgBR may be essential in promoting ER-positive tumor cell proliferation via survivin induction in breast cancer.     

Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation

Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions

H. P. Dong, A. K. Ree Rosnes, A. J. Bock, A. Holth, V. A. Flørenes, C. G. Trope’, B. Risberg and B. Davidson Flow cytometric measurement of cellular FLICE‐inhibitory protein (c‐FLIP) in ovarian carcinoma effusions Objective: The objective of this study was to establish a flow cytometry assay for measuring c‐FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. Methods: Two c‐FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c‐FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal‐to‐noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c‐FLIP expression was analysed for association with clinicopathological parameters and survival. Results: Rabbit polyclonal c‐FLIP by Abcam and the IntraStain kit by Dako performed best. c‐FLIP expression was detected in tumour cells in all 69 effusions (expression range 21–100%, median = 80%). No association was found between c‐FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c‐FLIP levels and expression of the previously studied apoptosis marker cleaved caspase‐3 (P = 0.029). Conclusions: An assay for measuring c‐FLIP in cytology specimens is presented. c‐FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.     

A two‐component enzyme complex is required for dolichol biosynthesis in tomato

Dolichol plays an indispensable role in the N‐glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a ‘glycan’, which is preassembled onto a membrane‐anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis‐prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi‐mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a ~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT‐binding protein (SlCPTBP), which is a close homolog of the human Nogo‐B receptor. Yeast two‐hybrid and co‐immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact in vivo and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2?. Co‐expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the ER, the site of dolichol accumulation and synthesis in eukaryotes.     

Vascular cell lines expressing SSAO/VAP-1: a new experimental tool to study its involvement in vascular diseases

Background information. PrAO (primary amine oxidase), also known as SSAO (semicarbazide‐sensitive amine oxidase)/VAP‐1 (vascular adhesion protein‐1), is an enzyme (EC 1.4.3.21) that is highly expressed in blood vessels and participates in many cell processes, including glucose handling or inflammatory leucocyte recruitment. High activity levels of this enzyme are associated with diabetes, atherosclerosis, AD (Alzheimer's disease) or stroke, among others, thus meaning that studies concerning SSAO as a therapeutic target are becoming more frequent. However, the study of this enzyme is difficult, owing to its loss of expression in cell cultures. Results. We have developed an endothelial cell line that stably expresses the human SSAO/VAP‐1 to be used as endothelial cell model for the study of this enzyme. The transfected protein is mainly expressed as a dimer in the membrane of these cells, and we demonstrate its specific localization in the lipid rafts of endothelial cells. The protein shows levels of enzymatic activity and kinetic parameters comparable with those observed in vivo by the same cell type. The transfected SSAO/VAP‐1 is also able to mediate the adhesion of leucocytes to the endothelium, a known function of this protein under inflammatory conditions. This distinctive function is not exerted by the SSAO/VAP‐1 transfected protein in a smooth muscle cell line that expresses 3‐fold higher protein levels. These differences have been widely reported to exist in vivo. Furthermore, using this endothelial cell model, we describe for the first time the involvement of the leucocyte‐adhesion activity of SSAO/VAP‐1 in the Aβ (amyloid β‐peptide)‐mediated pro‐inflammatory effect. Conclusions. The characterization of this new cell line shows the correct behaviour of the transfected protein and endorses the use of these cellular models for the in‐depth study of the currently poorly understood functions of SSAO/VAP‐1 and its involvement in the above‐mentioned pathologies. This cellular model will be also useful for the evaluation of potential compounds that could modulate its activity for therapeutic purposes.     

Influence of Klotho gene polymorphisms on vascular gene expression and its relationship to cardiovascular disease

Klotho protein has been associated with beneficial effects that contribute to the maintenance of cardiovascular health. Diverse studies suggest that alterations in the levels of this molecule may be associated with pathophysiological abnormalities that result in increased cardiovascular risk. The primary aim of this proof‐of‐concept study was to analyse the existence of a potential link between Klotho gene polymorphisms and the expression level of this gene in the vascular wall, and additionally with the incidence of cardiovascular disease and cardiovascular risk factors. Our results indicate that the variant G‐395A, located in the promoter region, influences Klotho gene vascular expression and is associated with the incidence of diabetes. Similarly, the exonic variant KL‐VS was associated with the incidence of atherosclerotic vascular disease and coronary artery disease. Moreover, vascular expression levels of Klotho were related with the incidence of diabetes mellitus and coronary artery disease. These findings, which need to be confirmed in larger studies, suggest a potential role of Klotho in the pathogenesis of vascular damage.     

The novel protein MANI modulates neurogenesis and neurite‐cone growth

Neuronal regeneration and axonal re‐growth in the injured mammalian central nervous system remains an unsolved field. To date, three myelin‐associated proteins [Nogo or reticulon 4 (RTN4), myelin‐associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG)] are known to inhibit axonal regeneration via activation of the neuronal glycosylphosphatidylinositol‐anchored Nogo receptor [NgR, together with p75 neurotrophin receptor (p75NTR) and Lingo‐1]. In the present study we describe the novel protein MANI (myelin‐associated neurite‐outgrowth inhibitor) that localizes to neural membranes. Functional characterization of MANI overexpressing neural stem cells (NSCs) revealed that the protein promotes differentiation into catecholaminergic neurons. Yeast two‐hybrid screening and co‐immunoprecipitation experiments confirmed the cell division cycle protein 27 (Cdc27) as an interacting partner of Mani. The analyses of Mani‐overexpressing PC12 cells demonstrated that Mani retards neuronal axonal growth as a positive effector of Cdc27 expression and activity. We show that knockdown of Cdc27, a component of the anaphase‐promoting complex (APC), leads to enhanced neurite outgrowth. Our finding describes the novel MANI‐Cdc27‐APC pathway as an important cascade that prevents neurons from extending axons, thus providing implications for the potential treatment of neurodegenerative diseases.     

Adipogenic and Antiapoptotic Protein Levels in Human Adipose Stromal Cells after Weight Loss

Objective: Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. However, current strategies to achieve sustained weight loss are often unsuccessful. Fat reaccumulation might be favored by enhanced adipose cell differentiation or survival in the postreduced state. Research Methods and Procedures: We measured adipogenic and apoptotic protein expression in subcutaneous abdominal adipose stromal‐vascular cells from 10 obese patients (7 women and 3 men) that were obtained before and after a 16% weight loss in a medically supervised weight loss program. Results: After weight loss, protein expression was 2.4‐fold higher (p < 0.005) for p42 C/CAAT enhancer binding protein α, but there was no change for peroxisome proliferator‐activated receptor γ1; both of these are adipogenic regulators. For neuronal apoptosis inhibitory protein, a protein associated with adipose cell apoptotic resistance, there was a rise of 1.7‐fold (p < 0.02). Discussion: Alterations in C/CAAT enhancer binding protein α and neuronal apoptosis inhibitory protein expression occurred in human adipose stromal‐vascular cells after weight loss in a pilot study of 10 patients. It will be important for future studies to directly examine whether the adipogenic and antiapoptotic capacity of these cells is changed after weight loss.     

Bacterial glycosyltransferase toxins

Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.     

Expression of apurinic/apyrimidinic endonuclease-1 (APE-1) in H. pylori-associated gastritis, gastric adenoma, and gastric cancer

Background and Aim: Apurinic/apyrimidinic endonuclease‐1 (APE‐1) is a key enzyme in DNA base excision repair (BER), linked to cancer chemosensitivity. However, little is known about the localization of APE‐1 in Helicobacter pylori‐infected gastric mucosa or its role in the development of gastric cancer. To investigate the role of APE‐1 in the development of gastric cancer, we examined APE‐1 expression and localization in cultured cells and gastric biopsies from patients with H. pylori‐infected gastritis or gastric adenoma, and from surgically resected gastric cancer. Methods: APE‐1 mRNA and protein expression were determined in H. pylori (CagA+) water‐extract protein (HPWEP)‐stimulated MKN‐28 cells, gastric adenocarcinoma cell‐line (AGS) cells, and human peripheral macrophages by real‐time polymerase chain reaction and Western blot analysis. APE‐1 expression and 8‐OHdG as a measure of oxidative DNA damage were evaluated by immunostaining. Localization of APE‐1 and IκBα phosphorylation in gastric adenoma and gastric cancer tissues were evaluated by single‐ and double‐label immunohistochemistry. Results: In studies in vitro, HPWEP‐stimulation significantly increased APE‐1 mRNA expression levels in both MKN‐28 cells and human peripheral macrophages. Hypo/reoxygenation treatment significantly increased APE‐1 protein expression in HPWEP‐stimulated MKN‐28 cells. HPWEP stimulation significantly increased both APE‐1 expression and IκBα phosphorylation levels in MKN‐28 and AGS cells. In human tissues, APE‐1 expression in H. pylori‐infected gastritis without goblet cell metaplasia was significantly increased as compared to that in tissues from uninfected subjects. Eradication therapy significantly reduced both APE‐1 and 8‐OHdG expression levels in the gastric mucosa. APE‐1 expression was mainly localized in epithelial cells within gastric adenoma and in mesenchymal cells of gastric cancer tissues. APE‐1 expression in gastric cancer tissues was significantly reduced compared to that in H. pylori‐infected gastric adenoma, while 8‐OHdG index and IκBα phosphorylation levels did not differ between these two neoplastic tissue types. Co‐localization of APE‐1 and IκBα phosphorylation was observed not in gastric cancer cells but in gastric adenoma cells. Conclusion: H. pylori infection is associated with increased APE‐1 expression in human cell lines and in gastric tissues from subjects with gastritis and gastric adenomas. The observed distinct expression patterns of APE‐1 and 8‐OHdG in gastric adenoma and gastric cancer tissues may provide insight into the progression of these conditions and warrants further investigation.     

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.     

Distinct donor and acceptor specificities of Trypanosoma brucei oligosaccharyltransferases

Asparagine‐linked glycosylation is catalysed by oligosaccharyltransferase (OTase). In Trypanosoma brucei OTase activity is catalysed by single‐subunit enzymes encoded by three paralogous genes of which TbSTT3B and TbSTT3C can complement a yeast Δstt3 mutant. The two enzymes have overlapping but distinct peptide acceptor specificities, with TbSTT3C displaying an enhanced ability to glycosylate sites flanked by acidic residues. TbSTT3A and TbSTT3B, but not TbSTT3C, are transcribed in the bloodstream and procyclic life cycle stages of T. brucei. Selective knockdown and analysis of parasite protein N‐glycosylation showed that TbSTT3A selectively transfers biantennary Man₅GlcNAc₂ to specific glycosylation sites whereas TbSTT3B selectively transfers triantennary Man₉GlcNAc₂ to others. Analysis of T. brucei glycosylation site occupancy showed that TbSTT3A and TbSTT3B glycosylate sites in acidic to neutral and neutral to basic regions of polypeptide, respectively. This embodiment of distinct specificities in single‐subunit OTases may have implications for recombinant glycoprotein engineering. TbSTT3A and TbSTT3B could be knocked down individually, but not collectively, in tissue culture. However, both were independently essential for parasite growth in mice, suggesting that inhibiting protein N‐glycosylation could have therapeutic potential against trypanosomiasis.     

PINCH: More than just an adaptor protein in cellular response

Particularly interesting new cysteine‐histidine‐rich protein (PINCH) is a LIM‐domain‐only adaptor protein involved in protein recruitment, subsequent assembly of multi‐protein complexes, and subcellular localization of these complexes. PINCH is developmentally regulated and its expression is critical for proper cytoskeletal organization and extracellular matrix adhesion. Although PINCH has no catalytic abilities, the PIP (PINCH–ILK–parvin) complex serves as a link between integrins and components of growth factor receptor kinase and GTPase signaling pathways. Accordingly, PINCH‐mediated signaling induces cell migration, spreading, and survival. Further research on the signaling cascades affected by PINCH is key to appreciating its biological significance in cell fate and systems maintenance, as the developmental functions of PINCH may extend to disease states and the cellular response to damage. PINCH is implicated in a diverse array of diseases including renal failure, cardiomyopathy, nervous system degeneration and demyelination, and tumorigenesis. This review presents evidence for PINCH's structural and functional importance in normal cellular processes and in pathogenesis. The current data for PINCH expression in nervous system disease is substantial, but due to the complex and ubiquitous nature of this protein, our understanding of its function in pathology remains unclear. In this review, an overview of studies identifying PINCH binding partners, their molecular interactions, and the potentially overlapping role(s) of PINCH in cancer and in nervous system diseases will be discussed. Many questions remain regarding PINCH's role in cells. What induces cell‐specific PINCH expression? How does PINCH expression contribute to cell fate in the central nervous system? More broadly, is PINCH expression in disease a good thing? Clarifying the ambiguous functions of PINCH expression in the central nervous system and other systems is important to understand more clearly signaling events both in health and disease. J. Cell. Physiol. 226: 940–947, 2011. © 2010 Wiley‐Liss, Inc.