Specific Regulatory Protein Research Articles

The Role of Hypoxia-Induced Factor in Cell Metabolism

Objective. Analysis and synthesis of literature data on morphological and functional properties and the diagnostic value of induced factor hypoxia. Methods. The basis of this study was a review of literature on this topic. Results. An important role in the adaptation of the organism to hypoxia belongs to a specific regulatory protein - hypoxia-induced factor (HIF), the activity of which increases with decreasing oxygen tension in the blood. HIF is a heterodimeric protein, the beta subunit of which is constantly expressed, and the synthesis of the alpha subunit is regulated by oxygen. Conclusion. Acute oxygen deficiency is the basis of a variety of pathological processes in many diseases and environmental factors. The hypoxia-induced factor is responsible for the formation of long-term adaptation to hypoxia, and therefore is a suitable target for pharmacological effects. The search for drugs that act as inducers or inhibitors of its synthesis is an important area in experimental pharmacology, since it allows not only to regulate the processes of adaptation to hypoxia, but more effectively treat cerebrovascular, cardiovascular, oncological and other diseases in whose genesis the leading role plays oxygen deficiency.

Angiopoietin-2 regulates vessels encapsulated by tumor clusters positive hepatocellular carcinoma nest-type metastasis via integrin α5β1

Objective: To investigate the molecular mechanism of nest metastasis in blood vessels encapsulated by tumor clusters (VETC) positive hepatocellular carcinoma (HCC). Methods: A total of 72 paraffin embedded HCC tissue samples were collected. Immunohistochemistry staining with CD34 (vascular endothelial cell marker protein) was used to observe the morphological manifestations of VETC cancer nests in primary tumors, bile duct cancerous thrombi and portal vein cancerous thrombi, and to study the characteristics of hematogenous metastasis of VETC cancer nests. Bioinformatics was used to predict the key proteins closely related to VETC cancer nest formation. Immunohistochemistry was used to detect the expression of angiogenin-2 (Ang-2), integrin α5, Integrin β1, and cyclooxygenase-2 (COX-2) proteins in HCC. Transwell cell migration assay was used to detect the effect of Ang-2/integrin α5β1 protein on the migration ability of endothelial cells and HCC cells. Western blotting was used to detect the effect of Ang-2/integrin α5β1 protein on the activity of focal adhesion kinase (FAK) protein. Results: Of the collected HCC specimens, 27 cases (27/72) were VETC (+), including 3 cases with biliary duct cancerous thrombus, 5 cases with portal vein cancerous thrombus, and 3 cases with both biliary duct cancerous thrombus and portal vein cancerous thrombus. VETC (+) HCC could metastasize to portal vein, bile duct, and liver in the form of cancer nest, and the nests retain their intact structure. Ang-2, integrin α5 and integrin β1 were overexpressed in tumor cells and endothelial cells of VETC (+) HCC nests, while COX-2 was only overexpressed in tumor cells of VETC (+) HCC nest. Ang-2 could promote the migration of HCC cell [(121±12) vs (186±11), P<0.01] and endothelial cells [(81±7) vs (163±14), P<0.01]. Integrin α5β1 activation antagonist ATN-161 could significantly block the ability of Ang-2 to promote the migration of HCC cells [(185±10) vs (135±9), P<0.05] and endothelial cells [(156±14) vs (103±6), P<0.05]. ATN-161 could significantly block the phosphorylation of FAK in HCC and endothelial cells induced by Ang-2. Conclusions: VETC (+) HCC could metastasize as a whole in a nested form, and possesses a specific regulatory protein. Ang-2/α5β1/FAK might be potential protein targets in the treatment of VETC (+) HCC nest-type metastasis.

IMPLICATION OF TROPONIN I RELEASE IN TRAUMA PATIENTS WITH HIP FRACTURES

Cardiac troponin I (TnI) is a cardiac specific regulatory protein that has been proven to be important in tracking myocardial cell injury and future mortality. Studies have proposed increases in TnI can often identify cardiovascular (CV) complications that correlate with increased mortality for non-

Gonadotropin releasing hormone activation of the mTORC2/Rictor complex regulates actin remodeling and ERK activity in LβT2 cells

The mammalian target of rapamycin (mTOR) assembles into two different multi-protein complexes, mTORC1 and mTORC2. The mTORC2 complex is distinct due to the unique expression of the specific core regulatory protein Rictor (rapamycin-insensitive companion of mTOR). mTORC2 has been implicated in regulating actin cytoskeletal reorganization but its role in gonadotrope function is unknown. Using the gonadotrope-derived LβT2 cell line, we find that the GnRH agonist buserelin (GnRHa) phosphorylates both mTOR and Rictor. Interestingly, inhibition of mTORC2 blunts GnRHa-induced cyto-architectural rearrangements. Coincident with blunting of actin reorganization, inhibition of mTORC2 also attenuates GnRHa-mediated activation of both protein kinase C (PKC) and extracellular signal regulated kinase (ERK). Collectively, our data suggests that GnRHa-mediated mTORC2 activation is important in facilitating actin reorganization events critical for initiating PKC activity and subsequent ERK phosphorylation in the gonadotrope-derived LβT2 cell line.

Scientists Explore a Master Genetic Switch That Plays a Key Role in Energy Metabolism and Human Brain Evolution and Function

Scientists have long used comparative animal studies to better understand the nuances of human evolution, from making diverse body plans to the emergence of entirely powerful and unique features structures, including the human brain. Now, in a new study appearing in the advanced online edition of Molecular Biology and Evolution, corresponding authors Perdomo-Sabogal et al. (2016) have explored the global gene regulator GABPa to better understand its influence as a master switch. Their work honed in on human-specific DNA regions potentially contributing to human evolution in key functions including cell energy, division and death, and diseases ranging from brain disorders to diabetes. Many of the GABPa gene regulatory sites were found to be significantly enriched at genes important for brain, central nervous system and spinal cord functions that are unique to humans, an area ripe for future research, and GABPa deficits linked to Alzheimer’s and Parkinson diseases, breast cancer, and autism. GABPa exerts its profound genomic influence by a tandem repeat of four highly conserved DNA letters. The GGAA consensus motif is found to control genes in all primates, mice, dogs, and cows, underscoring its importance in these animals. By performing ChIP-Seq experiments in a human cell line, the group found that GABPa exercises its influence across the genome at 11,619 putative GABPa binding sites representing nearly 4,000 genes. Through a tour de force sequence comparison of the human GABPa binding sites with similar sequences from 34 mammals, they identified 224 GABPa binding sites unique to humans. Comparing chimpanzee, macaque, and human cell lines and mutating the GGAA sequence to more ancestral states to determine how these changes affected GABPa function, they further narrowed down their function. For instance, when the human GABPa binding sites were introduced into primate cells, gene expression was increased up to five times in 17 out of 18 cases, indicating that a recent human mutation in GABPa resulted in higher gene expression and forces behind the evolution of novel, uniquely human functions. “Mutations that cause changes in the regulation of gene activities are one of the major factors in shaping species during evolution,” said corresponding author Robert Querfurth. “Our study demonstrates how, out of the millions of DNA regions, in which we differ from other apes, we can sift out those that, in response to a specific regulatory protein, cause gene activity changes in human cells. In this set, we find genes involved in brain and breast development and also in diseases like Alzheimer’s and Parkinson’s.” Reference

First Genome-Wide Mutational Map of Macaques a Boon for Evolutionary Studies.

Scientists have long used comparative animal studies to better understand the nuances of human evolution, from making diverse body plans to the emergence of entirely powerful and unique features structures, including the human brain. Now, in a new study appearing in the advanced online edition of Molecular Biology and Evolution, corresponding authors Perdomo-Sabogal et al. (2016) have explored the global gene regulator GABPa to better understand its influence as a master switch. Their work honed in on human-specific DNA regions potentially contributing to human evolution in key functions including cell energy, division and death, and diseases ranging from brain disorders to diabetes. Many of the GABPa gene regulatory sites were found to be significantly enriched at genes important for brain, central nervous system and spinal cord functions that are unique to humans, an area ripe for future research, and GABPa deficits linked to Alzheimer’s and Parkinson diseases, breast cancer, and autism. GABPa exerts its profound genomic influence by a tandem repeat of four highly conserved DNA letters. The GGAA consensus motif is found to control genes in all primates, mice, dogs, and cows, underscoring its importance in these animals. By performing ChIP-Seq experiments in a human cell line, the group found that GABPa exercises its influence across the genome at 11,619 putative GABPa binding sites representing nearly 4,000 genes. Through a tour de force sequence comparison of the human GABPa binding sites with similar sequences from 34 mammals, they identified 224 GABPa binding sites unique to humans. Comparing chimpanzee, macaque, and human cell lines and mutating the GGAA sequence to more ancestral states to determine how these changes affected GABPa function, they further narrowed down their function. For instance, when the human GABPa binding sites were introduced into primate cells, gene expression was increased up to five times in 17 out of 18 cases, indicating that a recent human mutation in GABPa resulted in higher gene expression and forces behind the evolution of novel, uniquely human functions. “Mutations that cause changes in the regulation of gene activities are one of the major factors in shaping species during evolution,” said corresponding author Robert Querfurth. “Our study demonstrates how, out of the millions of DNA regions, in which we differ from other apes, we can sift out those that, in response to a specific regulatory protein, cause gene activity changes in human cells. In this set, we find genes involved in brain and breast development and also in diseases like Alzheimer’s and Parkinson’s.”

Characterization of two MODY2 mutations with different susceptibility to activation

Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation.

INDUCERS OF THE REGULATORY FACTOR TO HYPOXIA ADAPTATION

The review is devoted to the analysis of the modern conceptions about role of specific regulatory protein HIF-1α (hypoxia-inducible factor-1alpha) in the mechanisms of adaptation to hypoxia. Experimental and clinical results of using of different HIF-1α inducers (inhibitors of HIF-1α degradation and inactivation, activators of transcription and translation of HIF-1α) to activate the processes of immediate and delayed organism adaptation to hypoxia is discussed in the article. This approach opens promising opportunities for effective pharmacotherapy of cardiovascular and other diseases with hypoxia and ischemia in their pathogenesis.

CDK2-AP1 inhibits growth of breast cancer cells by regulating cell cycle and increasing docetaxel sensitivity in vivo and in vitro.

BackgroundCell cycle regulatory pathway is a well-established pathway mainly dependent on cyclin-dependent kinases (CDKs), which are regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors(CKIs). Cyclin-dependent kinase 2 associate protein 1(CDK2-AP1) is a specific negative regulatory protein for CDK2, is important in the cancer cell cycle. However, the function of CDK2-AP1 in breast cancer remains unclear. We designed therefore explored the effects of CDK2-AP1 on breast cancer growth and its chemo-sensitivity.MethodsExpression of CDK2-AP1, CDK2 and CyclinD1 in 209 cases of pathological specimens using IHC staining was measured. Lost-of-function and Gain-of-function assays were used in vivo and in vitro relating to the specific role of CDK2-AP1 in breast cancer. We analyzed in vivo and in vitro the impact of CDK2-AP1 on chemotherapy sensitivity in breast cancer.ResultsThe positive ratio of CDK2-AP1 expression was reduced successively in normal breast tissue, DCIS, invasive breast cancer and relapsed breast cancer, however, with CDK2 and CyclinD1 it was suggested that CDK2-AP1 was correlated closely with the tumorigenesis and progress, and might work as a tumor suppressor. After down-regulating CDK2-AP1 in breast cancer cells, the cell cycle was accelerated and cell proliferation enhanced. The cell cycle was arrested in G0/G1 phase and G2/M phase after up-regulating CDK2-AP1 in breast cancer cells, inhibiting cell proliferation. The expression of CDK2 and CyclinD1 changed accordingly after downregulation or upregulation of CDK2-AP1 by western blot, suggesting a role of the CDK2-AP1/CDK2/CyclinD1 cell cycle pathway in the initiation and progression of breast cancer. Similar results were obtained in animal assays. The data indicates that CDK2-AP1 can induce sensitivity to docetaxel treatment in breast cancer cells.ConclusionsCDK2-AP1 affects tumorigenesis, tumor growth and chemo-sensitivity by cell cycle regulation, which can potentially to be a therapeutical agent in breast cancer.

Calponin levels in term laboring women

Objective: Calponin, a specific smooth muscle contraction regulatory troponin-like protein, is present in large quantities in uterine smooth muscle. Serum troponin levels rise in acute myocardial infarction, and creatine phosphokinase levels rise at high physical activity, both due to destruction of cardiac and striated muscle fibers. We hypothesize that the active labor process may cause uterine smooth muscle cell damage, which may result in rising maternal serum calponin levels. This was a preliminary study, searching for a new biomarker for preterm labor.Methods: The study group included laboring term primiparous women with a singleton fetus. The control group included similar demographic and pregnancy characteristics pregnant women not in labor. Maternal serum levels of calponin basic isoform were measured evaluated and compared in both groups.Results: Study group included 100 pregnant women. Calponin serum levels were higher in the active labor (794 ± 974 ng/mL) than in the group not in labor (591 ± 587 ng/mL), although it did not reach statistical significance. Gender and neonatal weight were similar in the two study groups.Conclusions: Calponin serum levels showed moderate elevation during active labor, compared to the levels in a cohort of pregnant comparable women at the same gestational weeks but not in labor.

Highlights from Recent Cancer Literature

Schramek D, Sendoel A, Segal JP, Beronja S, Heller E, Oristian D, et al. Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas. Science 2014;343:309–13.Schramek and colleagues address the relevance of passenger mutations to oncogenesis by utilizing ultrasound-guided lentiviral shRNA delivery to infect single-layer embryonic day-9.5 ectoderm. Pools of shRNA targeting genes relevant to head and neck squamous cell carcinomas (HNSCC) were introduced into the ectoderm of mice sensitized to HNSCC development; all showed increased numbers of HNSCCs. Forty percent of the tumors were enriched for shRNAs against Myh9, nonmuscle myosin IIa. Cells lacking Myh9 expression were defective for biologic endpoints associated with the ATPase domain of MYH9, acting independently of actin polymerization. These findings suggest that MYH9 suppresses tumors by stabilizing p53 in the nucleus.Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D, et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science 2013;342:971–6.Iida N, Dzutsev A, Stewart CA, Smith L, Bouladoux N, Weingarten RA, et al. Commensal bacteria control cancer response to therapy by modulating the tumor microenvironmnent. Science 2013;342:967–70.Two recent studies published in Science suggest that commensal intestinal bacteria influence the outcome of cancer treatment. Using mouse transplantable cancer models, Viaud and colleagues showed that efficacy of cyclophosphamide depends on “normal healthy” gut microbiota, whereas Iida and colleagues report that subcutaneous mouse tumors did not respond to platinum chemotherapy or immunotherapy after antibiotic therapy. In both cases, gut bacteria normally prime the tumor-associated leukocytes to a more effective immune response after therapy. Antibiotics are used prophylactically in some cancer patients. These data suggest that manipulation of the gut microbiome influences outcome, and that commensal microbes are critical when developing or assessing treatments. After all, 90% of our cells are not human.Fantozzi A, Gruber DC, Pisarsky L, Heck C, Kunita A, Yilmaz M, et al. VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation. Cancer Res; Published OnlineFirst January 10, 2014; doi:10.1158/0008-5472.CAN-13-1641.Epithelial-to-mesenchymal transition (EMT) enables invasion, metastases, and cancer stem cells. To clarify molecular mechanisms, Fantozzi and colleagues demonstrated that EMT upregulated expression of VEGFA and increased angiogenesis, enhancing tumorigenicity of murine breast cancer cells. Whereas expression of VEGFA was necessary for increased tumor initiation in cells that had undergone EMT, VEGFA itself was unable to support tumor formation of epithelial breast cancer cells before EMT, indicating the requirement for additional factors induced during EMT. The authors propose that the connecting mechanism between cancer cell stemness and tumor initiation might be cancer stem cells with EMT-induced, VEGFA-mediated angiogenesis.White AC, Khuu JK, Dang CY, Hu J, Tran KV, Liu A, Gomez S, Zhang Z, Yi R, Scumpia P, Grigorian M, Lowry WE. Stem cell quiescence acts as a tumour suppressor in squamous tumours. Nat Cell Biol 2014;16:99–107.How does oncogenic signaling take place in quiescent adult stem cells? White and colleagues show that hair follicle stem cells (HFSC) exist in two states: telogen when quiescent, and anagen when proliferative. HFSCs are insulated from oncogenic signaling during quiescence (telogen) and can be RasG12D transformed only when activated into anagen. During quiescence, PTEN activation and loss/inhibition of AKT, CREB, and JUN made HFSCs refractory to Ras. Pten loss downregulated genes that induced telogen (i.e., FGF18 and BMP2/4/6), but did not affect genes that induced anagen. Thus, loss of Pten created a telogen phase sensitive to tumor initiation. These data suggest that quiescence is dominant over oncogenic signaling, and that specific cues may reprogram malignant cells into quiescence despite powerful oncogenes.Lubanska D, Market-Velker BA, Decarvalho AC, Mikkelsen T, Fidalgo da Silva E, Porter LA. The cyclin-like protein Spy1 regulates growth and division characteristics of the CD133+ population in human glioma. Cancer Cell 2014;25:64–76.To characterize mechanisms related to stemness in glioma-initiating cells (GIC), Lubanska and colleagues studied the cyclin-like protein, SPY1 (SPDYA). Amplification of SPY1 was correlated with poor prognosis in patients. Knockdown caused decreased proliferation, loss of GIC markers, increased expression of neuronal markers, and increased asymmetric division of GICs, whereas overexpression stabilized a stem-like state in neural stem cells. Interestingly, while loss of SPY1 blocked CDK2, knockdown of the CDK2 regulator cyclin E did not similarly affect stemness or asymmetric division. Overall, these findings demonstrate a role for a specific cell-cycle regulatory protein in balancing cell division and stemness in glioma.Note: Breaking Advances are written by Cancer Research editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.

Hypoxia-inducible factor as a pharmacological target

The review is devoted to the role of specific regulatory protein HIF-1α (hypoxia-induced factor-1alpha) in the mechanisms of the organism adaptation to hypoxia. Pharmacological influence on HIF-1α for a target upregulation of processes of immediate and delayed organism adaptation to hypoxia is discussed in the article. Such approach opens new possibilities of effective pharmacotherapy of oncological, rheumatic, cardio-vascular and other diseases which have hypoxia and ischemia in their pathogenetic state.

Characterisation of FEZ1-mediated inhibition of retroviral replication

The brain specific cytoskeletal regulatory protein FEZ1 (fasciculation and elongation protein zeta 1) has been shown to block retroviral infection including human immunodeficiency virus type 1 (HIV-1). Previous work has shown that FEZ1 blocks HIV-1 early in the viral life cycle, after reverse transcription but before nuclear entry. To begin to dissect the mechanism by which FEZ1 functions in host cell resistance yeast-two-hybrid assays were performed to identify FEZ1 interacting proteins. Two of the interactors, talin1 and vinculin, were identified as strong candidates for potential involvement in FEZ1-mediated inhibition of retroviral infection due to their cytoskeletal regulatory functions. Talin 1 and vinculin directly interact with each other and are involved in the linkage of integrins to the actin cytoskeleton. To assess the potential role of talin1 and vinculin in retroviral infectivitiy we have used an siRNA approach to reduce expression of each gene followed by infection with pseudo-typed HIV-1 virus. Two independent infection assays confirmed that cells with reduced expression of either talin 1 or vinculin become more susceptible to HIV-1 infection. Interestingly, when expression of either the talin 1 or vinculin gene is reduced FEZ1 expression levels also decrease. We can conclude that FEZ1, talin 1 and vinculin play an important role in the HIV-1 lifecycle and we are currently examining whether these proteins function independently or as part of a larger protein complex that regulates cellular susceptibility to retroviral infection.

Glucokinase and molecular aspects of liver glycogen metabolism

Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.

Contribution of Peroxisome-specific Isoform of Lon Protease in Sorting PTS1 Proteins to Peroxisomes

Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in beta-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the beta-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.

Autotaxin and lysophosphatidic acid stimulate intestinal cell motility by redistribution of the actin modifying protein villin to the developing lamellipodia

Autotaxin (ATX) is a potent tumor cell motogen that can produce lysophosphatidic acid (LPA) from lysophosphatidylcholine. LPA is a lipid mediator that has also been shown to modulate tumor cell invasion. Autotaxin mRNA is expressed at significant levels in the intestine. Likewise, LPA 2 receptor levels have been shown to be elevated in colon cancers. The molecular mechanism of ATX/LPA-induced increase in intestinal cell migration however, remains poorly understood. Villin is an intestinal and renal epithelial cell specific actin regulatory protein that modifies epithelial cell migration. In this study we demonstrate that both Caco-2 (endogenous villin) and MDCK (exogenous villin) cells, which express primarily LPA 2 receptors, show enhanced cell migration in response to ATX/LPA. ATX and LPA treatment results in the rapid formation of lamellipodia and redistribution of villin to these cell surface structures, suggesting a role for villin in regulating this initial event of cell locomotion. The LPA-induced increase in cell migration required activation of c-src kinase and downstream tyrosine phosphorylation of villin by c-src kinase. LPA stimulated cell motility was determined to be insensitive to pertussis toxin, but was regulated by activation of PLC-γ 1. Together, our results show that in epithelial cells ATX and LPA act as strong stimulators of cell migration by recruiting PLC-γ 1 and villin, both of which participate in the initiation of protrusion.

Specific splicing regulatory protein human hTra2-beta but not hTra2-alpha is overexpressed in invasive human breast cancer

9621 Background: The two human dTra2 orthologs hTra2-alpha and hTra2-beta 1, members of the extended family of serine-argenine-rich (SR) splicingfactors, regulate alternative splicing of exons with...

Specific splicing regulatory protein human hTra2-beta but not hTra2-alpha is overexpressed in invasive human breast cancer

9621 Background: The two human dTra2 orthologs hTra2-alpha and hTra2-beta 1, members of the extended family of serine-argenine-rich (SR) splicingfactors, regulate alternative splicing of exons with...

Expression and initial promoter characterization of PCAN1 in retinal tissue and prostate cell lines.

Prostate cancer is the most frequently diagnosed neoplasia in men and one of the leading causes of cancer-related deaths in men over 60. In an effort to understand the molecular events leading to prostate cancer, we have identified PCAN1 (prostate cancer gene 1) (also known as GDEP), a gene that is highly expressed in prostate epithelial tissue and frequently mutated in prostate tumors. Here we demonstrate its expression in neural retina, and retinoblastoma cell culture but not retinal pigment epithelial cell culture. We further characterize PCAN1 expression in the prostate cell lines RWPE1, RWPE2, and LnCAP FGC. We demonstrate an increase in expression when the cells are grown in the presence of Matrigel, an artificial extracellular basement membrane. Expression was time dependent, with expression observed on d 6 and little or no expression on d 12. Testosterone was not found to increase PCAN1 expression in this culture system. In addition, normal prostate epithelial cells co-cultured with normal prostate stromal cells did not exhibit PCAN1 expression at any time. To definitively locate the transcription initiation sites, we performed restriction-ligase-mediated 5' RACE, to selectively amplify only mRNA with a 5' cap. An initial characterization of the sequence upstream of the initiation sites determined six possible binding sites for the prostate specific regulatory protein NKX3.1 and four potential binding sites for the PPAR/RXR heterodimer that is involved in the control of cell differentiation and apoptosis.

Identification of a soybean protein that interacts with GAGA element dinucleotide repeat DNA.

Dinucleotide repeat DNA with the pattern (GA)(n)/(TC)(n), so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)(n)/(CT)(n) DNA. Soybean GBP was dissimilar from the GAGA factor of Drosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)(n)/(CT)(n). GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum. In addition, Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants.