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Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.

Studies in yeast indicate that three specialized endoplasmic reticulum-associated degradation (ERAD) pathways, namely ERAD-L, -M, or -C, dispose substrates with structural lesions in the lumenal, transmembrane, or cytosolic domains, respectively. The ubiquitin ligase (E3) Hrd1p and its cooperating partners are required for ERAD-L and -M pathways, whereas Doa10p complex is required for the ERAD-C pathway. We investigated these pathways in mammalian cells by assessing the requirements of the mammalian ERAD E3s, gp78 and Hrd1, in degradation of four substrates each with different type of structural lesions: CD3δ, Z-variant α1-antitrypsin, tyrosinase (C89R) and mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508). We demonstrated that tyrosinase (C89R) is a substrate for Hrd1 while all others are gp78 substrates. Knockdown of Hrd1 diminished gp78 substrate levels, but silencing of gp78 had no effect on Hrd1's substrate, suggesting that the functional interaction between Hrd1 and gp78 is unidirectional. Furthermore, while Ufd1 is dispensable for gp78-mediated ERAD, it is essential for Hrd1-mediated ERAD. Interestingly, Npl4 was found to be a key component for both pathways. These results suggest that the Hrd1-mediated ERAD requires a well-established retrotranslocation machinery, the p97/VCP-Ufd1-Npl4 complex, whereas the gp78 pathway needs only p97/VCP and Npl4. In addition, the three distinct ERAD pathways described in yeast may not be strictly conserved in mammalian cells as gp78 can function on three substrates with different structural lesions., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The most common mutation associated with cystic fibrosis is the deletion of phenylalanine 508 of cystic fibrosis transmembrane conductance regulator (CFTRDeltaF508). This mutation renders otherwise functional protein susceptible to ER-associated degradation (ERAD) and prevents CFTR from exiting the ER and trafficking to the plasma membrane. In this study, we demonstrate that RNAi-mediated silencing of gp78, an established ubiquitin ligase (E3) involved in ERAD, leads to accumulation of CFTRDeltaF508 protein in cells. gp78 facilitates the degradation of CFTRDeltaF508 by enhancing both its ubiquitination and interaction with p97/VCP. SVIP, which is the inhibitor of gp78, causes accumulation of CFTRDeltaF508. We showed that endogenous gp78 co-immunoprecipitates with Hrd1. Furthermore, the results indicate that silencing the expression of another ERAD E3, Hrd1, leads to stabilization of gp78 and decline in gp78 ubiquitination; thereby enhancing CFTRDeltaF508 degradation. The results support that gp78 is an E3 targeting CFTRDeltaF508 for degradation and Hrd1 inhibits CFTRDeltaF508 degradation by acting as an E3 for gp78.

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.

Human liver CYP3A4 is an endoplasmic reticulum (ER)-anchored hemoprotein responsible for the metabolism of >50% of clinically prescribed drugs. After heterologous expression in Saccharomyces cerevisiae, it is degraded via the ubiquitin (Ub)-dependent 26S proteasomal pathway that utilizes Ubc7p/Cue1p, but none of the canonical Ub-ligases (E3s) Hrd1p/Hrd3p, Doa10p, and Rsp5p involved in ER-associated degradation (ERAD). To identify an Ub-ligase capable of ubiquitinating CYP3A4, we examined various in vitro reconstituted mammalian E3 systems, using purified and functionally characterized recombinant components. Of these, the cytosolic domain of the ER-protein gp78, also known as the tumor autocrine motility factor receptor (AMFR), an UBC7-dependent polytopic RING-finger E3, effectively ubiquitinated CYP3A4 in vitro, as did the UbcH5a-dependent cytosolic E3 CHIP. CYP3A4 immunoprecipitation coupled with anti-Ub immunoblotting analyses confirmed its ubiquitination in these reconstituted systems. Thus, both UBC7/gp78 and UbcH5a/CHIP may be involved in CYP3A4 ERAD, although their relative physiological contribution remains to be established.

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.

The receptor for the autocrine motility factor/phosphoglucose isomerase cytokine (gp78 or AMFR) has been extensively characterized using the 3F3A monoclonal antibody. Cloning of AMFR identified a seven-transmembrane domain G-protein-coupled receptor ubiquitin E3 ligase whose identity as AMFR was based on prior expression cloning with the 3F3A mAb that generated a truncated sequence. We show here that the gp78/AMFR gene product is indeed recognized by the 3F3A mAb. The FLAG-taggedAMFR immunoprecipitated with an anti-FLAG antibody was recognized by the 3F3A mAb in Western blot analysis and cells transfected with AMFR exhibit increased labeling with the 3F3A mAb. The 3F3A mAb does not however recognize higher molecular weight isoforms of AMFR. 3F3A labeling colocalizes with tagged AMFR in a peripheral ER network but does not recognize FLAG- or GFP-tagged AMFR localized to a perinuclear ER domain that likely corresponds to misfolded forms of the protein retained in the ER. These data indicate that 3F3A antibody binding is highly specific for a subpopulation of AMFR localized to an ER subdomain. Coexpression of AMFR-GFP and a lumenal ER-targeted RFP presented extensive colocalization in living cells andAMFR-GFP is concentrated in a basal ER network morphologically similar to that labeled by the 3F3A mAb in fixed cells. The3F3A anti-AMFR mAb therefore selectively recognizes a subpopulation of expressed AMFR localized to a subdomain of the ER.

Accumulation of ascites fluid often observed in some solid tumors is one of the most devastating conditions of a patient's difficulty in responding to treatment, and to a decrease in the quality of life. Various factors are thought to be associated with the formation of cancer-induced fluid accumulation and hyperpermeability of a blood vessel is thought to go with this process. Here, we report a new factor that is involved in this process, e.g., autocrine motility factor (AMF). AMF is a tumor-related cytokine which stimulates the tumor cell locomotion and migration and promotes tumor cell invasion during metastasis. AMF secretion and its receptor (AMFR) expression in tumor cells are closely correlated with disease aggravation of convalescence. The response of endothelial or mesothelial cellular morphological alternation to AMF leads to motile enhancement and vascular permeability. Tumor AMF induces gaps in an endothelial or mesothelial monolayer by stimulating a cellular movement, and accelerates the ascites accumulation. And treatment experiment with anti-AMF antibody succeeded in the reduction of the ascites accumulation, which renders AMF to the target molecule. It is suggested that AMF is one of the significant factors which relates to various pathological malignancies induced by tumor mass, and understanding of its function could benefit prognosis and treatment.

A tumour-secreted cytokine autocrine motility factor (AMF) induces in vivo invasion and metastasis, and in vitro tumour cell motility by a signal transduction through interaction with its cell surface receptor gp78. In this report, we investigated the characterization of a high-metastatic human oral squamous cell carcinoma (SCC) cell line LMF4 and low-metastatic HSC-3 in comparison with non-metastatic HSC-2 and HSC-4. Morphological and motility analyses revealed LMF4 cells to have the highest motile activity among those cells. However, LMF4 cells shared the similar features with HSC-3: high level secretion of AMF, enhancement of gp78 expression, co-expression of vimentin and cytokeratin, although LMF4 cells showed twice as high motile reactivity as HSC-3. The only difference was that LMF4 had twice as high amount of low-affinity receptor(s) as HSC-3, shown by Scatchard analysis.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment., (Copyright 2001 Academic Press.)

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment., (Copyright 2001 Academic Press.)

Background: Expression of autocrine motility factor receptor (AMFR) associates with increased cell migration and poor survival in certain types of human cancers. We assessed the possible correlation between AMFR, clinicopathologic features, and survival in stage I non-small cell lung cancer (NSCLC)., Methods: AMFR expression was analyzed immunohistochemically, using a monoclonal antibody (3F3A) in tumor specimens from 97 patients with curative resection. Vascular endothelial growth factor (VEGF) expression was also examined after accounting for AMFR expression., Results: Out of 97 tumors, 38 (39.2%) were positively stained with AMFR. The AMFR expression was significantly associated with histologic type of tumor, mainly in adenocarcinoma. Overall survival of patients with AMFR-positive tumors was significantly worse than that of AMFR-negative tumors (p = 0.0050). The AMFR expression appears to be associated with VEGF expression. Patients who were AMFR positive and had high VEGF expression had a worse prognosis compared with the AMFR-negative and low VEGF-expression group (p < 0.0001). Multivariate analysis revealed an independent prognostic impact of AMFR on survival (p = 0.0039)., Conclusions: These results indicate that evaluation of AMFR expression may provide useful guidance in follow-up of patients with NSCLC.

Urothelial carcinomas are heterogeneous diseases with an aggressive clinical potential. To date, the most used prognostic factors for bladder carcinomas are grade and stage, which are based on histopathological parameters that are often not reliable in predicting a clinical outcome. Here, we evaluated the clinical outcome of 100 patients with urothelial carcinomas with follow-up information for more than 2 years after surgery in relation to the expression of two cell surface antigens, ie, E-cadherin and autocrine motility factor receptor (AMF-R, gp78). Frozen bladder tissues were serially cut, stained either with hematoxylin and eosin for grading, with the anti-gp78 antibodies, or with anti-E-cadherin antibodies to determine level of expression. Of 63 patients presented at the time of diagnosis with pathological loss of E-cadherin associated with increased gp78 expression, 39 (62%) succumbed to tumor progression or death. Of 37 patients with normal E-cadherin and gp78 expression positive and negative, respectively, 36 (97%) had favorable disease outcomes (P < 0.0001). The results suggest that in bladder carcinomas abnormal expression of both E-cadherin and gp78 results in a poor disease outcome, independent of tumor stage and grade.

Autocrine motility factor (AMF) is a tumor-secreted cytokine that acts as a motogen as well as a mitogen via a receptor-mediated signaling pathway(s). Expression of the AMF receptor (AMF-R) in normal cells is regulated by cell contact whereas in transformed cells AMF-R is constitutively expressed irrespective of cell density. Here we have analyzed the regulation of AMF-R expression in a BALB/c angiosarcoma tumor system that allows investigation of cellular characteristics associated with tumor progression. The metastatic cell variant (A31-M) displayed a higher rate of unstimulated motility and responded to tumor-derived AMF locomotory stimulus as compared with the nonmetastatic cell variants (A31-TR and A31-TU) and, although a similar level of receptor expression was detected in cellular extracts from subconfluent cultures of these sublines, surface localization differed and cell contact down-regulated AMF-R expression in the normal but not the transformed cell counterparts. AMF promoted marked rearrangement of focal adhesion plaque proteins in the AMF migration-responsive cells exclusively. Reorganization of vinculin after AMF stimulation was paralleled by morphological redistribution of tyrosine-phosphorylated proteins and the tyrosine kinase pp125FAK in the migration-responsive cells; however, we did not observe a concomitant change in the pp125FAK phosphorylation state or the general level of cellular tyrosine phosphorylation in response to treatment, suggesting that the induction of cellular migration by AMF is independent of tyrosine phosphorylation events at the focal contacts and may therefore represent a novel pathway of cytokine-induced migration regulation.

Autocrine motility factor interacts with its cell surface receptor (AMF-R) to stimulate tumor cell motility. To study AMF-R expression following transformation of polarized epithelial MDCK cells, we have used the invasive Moloney sarcoma virus transformed MDCK (MSV-MDCK) cell population. Decreased E-cadherin expression of the transformed MSV-MDCK clones is associated with both increased cellular motility and increased AMF-R expression. Increased AMF-R expression is due to MSV transformation as differentially motile MSV-MDCK clones, which either retain low E-cadherin, exhibit equivalent high levels of AMF-R. Loss of the polarized epithelial phenotype and increased cellular motility following transformation of MDCK cells is thus associated with a shift from a high E-cadherin/low AMF-R to a low E-cadherin/low AMF-R phenotype.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

Autocrine motility factor (AMF) is a 55 kDa cytokine which is produced and secreted by cancer cells and which regulates cell motility via binding to its receptor, a 78 kDa cell surface glycoprotein (gp78-AMFR), and activating a pertussis toxin (PT)-sensitive G-protein. AMF purified from HT-1080 human fibrosarcoma cells stimulates the growth and motility of 3T3-A31-fibroblasts at a concentration of 0.1 ng/ml or less. The expression of total as well as cell surface gp78-AMFR is down-regulated in contact-inhibited A31-fibroblasts and AMF stimulates the healing of experimentally wounded, density-arrested A31 monolayer cultures. This is the first report of the paracrine and mitogenic actions of AMF and the results presented here show that AMF functions as a growth factor and suggest a possible role for its activity in normal tissue regeneration and tumor cell dissemination.