Expression Of PC2 Research Articles

P-104 production and glucose-dependent secretion of insulin by human spermatozoa

Abstract Study question Do human spermatozoa express insulin and the enzymes responsible for the cleavage of proinsulin? Is the secretion of insulin by spermatozoa responsive to glucose stimuli? Summary answer Human spermatozoa express insulin and PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. Spermatozoa produce and release insulin in a glucose concentration-dependent manner. What is known already Spermatogenesis is dependent on a finely regulated hormonal and metabolic control. Insulin is considered one of the most important regulators in the metabolic regulation of spermatogenesis, acting directly on the differentiation of spermatogonia into primary spermatocytes or indirectly through the metabolic modulation of both Sertoli and Leydig cells. In addition, insulin is hypothesized to play a major role in human spermatozoa capacitation, although the mechanisms that regulate its production and secretion remain to be disclosed. Study design, size, duration The main objective of this study was to estimate the capacity of insulin production by human spermatozoa and if they express PC1/3 and PC2, which are enzymes responsible for the cleavage of proinsulin. In addition, we hypothesized that insulin production could respond to glucose stimuli. Seminal samples of normozoospermic men (n = 15) seeking fertility treatment were collected for these experiments. Participants/materials, setting, methods Spermatozoa were submitted to a density gradient centrifugation and two fractions were collected according to their motility (high vs low motility). The expression of insulin, PC1/3 and PC2 mRNA was evaluated by RT-qPCR. The protein expression of insulin, PC1/3 and PC2 was evaluated by immunofluorescence. High motility spermatozoa were incubated in BWW medium with increasing glucose concentrations (0, 5.5, 11, and 22 mM). After 6 h, the concentration of secreted insulin was quantified by ELISA. Main results and the role of chance We were able to identify the expression of insulin, PC1/3, and PC2 mRNA, as well as the respective proteins, in human spermatozoa. Through immunofluorescence, we observed that insulin and both enzymes are mainly expressed in the midpiece. Human spermatozoa also express insulin receptor, mainly in the midpiece and tail. The mRNA expression of insulin and PC1/3 was found to be higher in the highly motile spermatozoa. Human spermatozoa produce very small quantities of insulin (pg/mL range), which suggests an autocrine or paracrine action. The release of insulin to the medium was found to occur in a glucose concentration-dependent manner. Limitations, reasons for caution This is an in vitro study with relatively small sample size. Future studies are required to further evaluate the role of insulin and its signalling pathway on human spermatozoa capacitation and fertilization capacity. Wider implications of the findings This study shows that human spermatozoa express PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. In addition, human spermatozoa release insulin in a glucose concentration-dependent manner. The study of insulin signalling opens the path for a deeper understanding of human spermatozoa bioenergetics and the development of novel therapies. Trial registration number Not applicable

Identification of Plasma Cell Antigen PC-1 as a Novel Cell Surface Marker of Germinal Center, Memory and Plasmacytoma B Cells.

The murine plasma cell (PC) alloantigen PC-1, also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), is expressed on PCs and by many non-lymphoid tissues. PC-1 is best known for its role in phosphate metabolism, hydrolyzing NTP to generate pyrophosphate. However, its expression and function in B lymphocytes remains unclear. Here we describe a previously unknown expression pattern of PC-1 in murine late stage B cells and B lymphomas. In normal mice, expression of PC-1, determined by flow cytometry, was very low on bone marrow pro-B, pre-B, immature B, and mature B cells, and on splenic follicular and marginal zone B cells. However, peritoneal B cells expressed significantly higher levels of PC-1 than splenic naïve B cells. CD5+ B1a cells expressed more PC-1 than B1b and B2 cells. Interestingly, PC-1 distinguishes two subsets of B1a: PC-1hi and PC-1lo. The PC-1hi B1a cells were IgMlo, B220lo, and larger in size while PC-1lo B1a cells were IgMhi, B220hi, and smaller in size. The PC-1hi B1a cells express higher levels of Blimp-1 transcripts than the PC-1lo B1a cells, indicating that the PC- 1hi B1a cells may exhibit features of pre-plasma cells. Furthermore, after immunization with NP-KLH, germinal center (GC) B cells, PCs and early memory B cells expressed 2–4-fold more PC-1 than naïve B cells. Naïve B cells stimulated with LPS or anti-IgM plus anti-CD40 antibodies also dramatically upregulated PC-1 expression. Finally, gene expression profiling studies of primary lymphomas in NFS.V(+) congenic mice revealed high expression levels of PC-1 in plasmacytomas, immunoblastic lymphomas, and centroblastic-follicular lymphoma types. These studies demonstrate that PC-1 can be a useful marker for identification of stages in GC and post-GC cell differentiation and of lymphomas with a GC or PC origin. The function of PC-1 in B cells is currently under investigation.

Polycystin-1 Downregulation Induces Aortic Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Dysfunction in Thoracic Aortic Dissection

Polycystin (PC)-1 is a transmembrane protein encoded by the gene of polycystic kidney disease-1 (PKD-1), which regulates cellular proliferation and migration. This study was designed to investigate the regulatory mechanisms of PC-1 on phenotypes of aortic vascular smooth muscle cells (VSMCs) and functions of extracellular matrix (ECM) in thoracic aortic dissection (TAD). In this work, aortic tissues from patients with TAD and healthy controls were collected, in vitro studies were also performed. Compared with the control group, expression of PC-1 and the contractile phenotypic markers of VSMCs were significantly decreased in TAD group, whereas expression of the synthetic markers of VSMCs, matrix metalloproteinase (MMP)-2, collagen I and collagen III were increased. The phosphorylation of mTOR, S6K and S6 were also elevated in TAD group. PC-1 downregulation of aortic VSMCs inhibited the expression of the contractile markers, but elevated the expression of the synthetic markers, MMP-2, collagen I and collagen III compared with the control group. The phosphorylation of mTOR, S6K and S6 were also increased in PKD-1-knockdwon VSMCs. PC-1 upregulation reversed all these expression characteristics in aortic VSMCs. Furthermore, rapamycin treatment to PKD-1-knockdwon VSMCs inhibited the effects caused by PC-1 downregulation. These results of our study have revealed that PC-1 downregulation induces aortic VSMCs phenotypic alteration and ECM dysfunction via activation of mTOR/S6K/S6 signaling pathway, thus the downregulation of PC-1 might be a potential mechanism for the development and progression of TAD. Funding Statement: This study was supported by National Natural Science Foundation of China (81700422 to Jing Zhang, 81570438, 81970412 to Li-xin Wang), Shanghai Science and Technology Foundation (14YF1412600 to Jing Zhang, 18441902400 to Li-xin Wang) and Shanghai Chest Hospital Project of Collaborative Innovation (YJXT20190207 to Jing Zhang). Declaration of Interests: The authors stated: None. Ethics Approval Statement: The collection and use of human aortic samples were approved by the Ethical Committee of Shanghai Chest Hospital.

Why don't corticotroph tumors always produce Cushing's disease?

Silent corticotroph tumors are a pituitary neuroendocrine tumor subtype of corticotroph lineage that do not clinically express Cushing's disease. The silencing of this type of tumor is not fully understood. The aim of the present study was to delve into the lack of secretory activity, studying the post-transcriptional and post-translational regulation of POMC/ACTH in a series of molecularly identified functioning and silent corticotroph tumors. We analyzed 24 silent corticotroph, 23 functioning corticotroph and 25 silent gonadotroph tumors. We used Sanger sequencing, quantitative real-time PCR and Western blot to analyze genetic alterations in POMC, gene expression of TBX19, NEUROD1, POMC, PCSK1, PCSK2, CPE and PAM and protein expression of POMC, PC1/3, PC2, CPE and PAM. We found different polymorphisms in the POMC gene of corticotroph tumors, some of them related to deficiency of proopiomelanocortin. Silent corticotroph tumors showed lower PC1/3 gene and protein expression than functioning ones, especially compared to micro-functioning corticotroph tumors (all P < 0.05). Moreover, we found a positive correlation between PC2 and CPE gene and protein expression (rho ≥ 0.670, P < 0.009) in silent corticotroph tumors compared with functioning ones. By studying the post-transcriptional and post-translational processing of POMC and ACTH, respectively, in a large series of silent and functioning corticotroph tumors, we found that the lack of secretory activity of these tumors is related to an impaired processing of POMC and a high degradation of ACTH, with the macro-functioning corticotroph tumor behaving as an intermediate state between micro-functioning and silent corticotroph tumors.

PARP-1 Inhibition Rescues Short Lifespan in Hyperglycemic C. Elegans And Improves GLP-1 Secretion in Human Cells.

TCF7L2 is located at one of the most strongly associated type 2 diabetes loci reported to date. We previously reported that the most abundant member of a specific protein complex to bind across the presumed causal variant at this locus, rs7903146, was poly [ADP-ribose] polymerase type 1 (PARP-1). We analyzed the impact of PARP-1 inhibition on C. elegans health in the setting of hyperglycemia and on glucose-stimulated GLP-1 secretion in human intestinal cells. Given that high glucose concentrations progressively shorten the lifespan of C. elegans, in part by impacting key well-conserved insulin-modulated signaling pathways, we investigated the effect of PARP-1 inhibition with Olaparib on the lifespan of C. elegans nematodes under varying hyperglycemic conditions. Subsequently, we investigated whether Olaparib treatment had any effect on glucose-stimulated GLP-1 secretion in the human NCI-H716 intestinal cell line, a model system for the investigation of enteroendocrine function. Treatment with 100uM Olaparib in nematodes exposed to high concentrations of glucose led to significant lifespan rescue. The beneficial lifespan effect of Olaparib appeared to require both PARP-1 and TCF7L2, since treatment had no effect in hyperglycemic conditions in knock-out worm strains for either of these homologs. Further investigation using the NCI-H716 cells revealed that Olaparib significantly enhanced secretion of the incretin, GLP-1, plus the gene expression of TCF7L2, GCG and PC1. These data from studies in both C. elegans and a human cell line suggest that PARP-1 inhibition offers a novel therapeutic avenue to treat type 2 diabetes.

Polycystin 2-dependent cardio-protective mechanisms revealed by cardiac stress.

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2+/- mice are hypersensitive to acute β-adrenergic receptor (βAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous βAR stimulus) on Pkd2+/- mice. After βAR stimulation for 7days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2+/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3days after cessation of the βAR stimulation, but not in Pkd2+/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2+/- mice and in ADPKD patients.

Posttranslational regulation of polycystin-2 protein expression as a novel mechanism of cholangiocyte reaction and repair from biliary damage.

Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)-mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2(-/-) knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2(-/-) mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2. In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases.

P1142 : Next generation sequencing analysis of cholestatic patients reveals high impact variation in common and rare candidate genes: Dissecting the genetic architecture of cholestasis

(Mdr2-KO, bile duct ligation, treatment with dehydrocollidine – DDC). However, PC-2-gene expression was not reduced, suggesting that the decrease in PC2 protein content was due to increased degradation. To understand if factors involved in biliary damage influence PC2 protein expression, mouse cholangiocytes were treated with pro-inflammatory cytokines (IL-1b, IFNg, TNFa), nitric oxide (NO) donors (DETAnonoate) and ER stressors (thapsigargin). Expression of PC2 protein was again significantly reduced, but not its gene expression. Noteworthy, downregulation of PC-2 was associated to increased ERK1/2 phosphorylation, HIF1a transcription activity, secretion of VEGF and phosphorylation of VEGFR2. Expression of Herp and NEK, ubiquitin-like proteins that promote PC2 degradation by increasing the retrotranslocation from the ER to the proteasome complex was also increased. Pre-treatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In the latter conditions, PC2 degradation was inhibited by LY294002, an inhibitor of PI3K that blocks the autophagosome formation and by blockade of lysosomes with chloroquine, suggesting a role for autophagy. Finally, treatment of DDC-treated mice with the proteasome inhibitor bortezomib, significantly reduced the extent of the liver damage and restored PC2 expression. Conclusions: PC2 protein is modulated post-translationally by proinflammatory cytokines, ER-stressors and NO-donors and is reduced in mice with biliary damage. Down regulation of PC2 protein expression in cholangiocytes increases the ERK1/2/HIF1a pathway and VEGF secretion, thereby playing a pivotal role in the regulation of cholangiocyte response to biliary damage. Treatments able to restore PC2 expression (i.e. proteasome inhibitors) may represent a new therapeutic approach in biliary diseases.

Tranexamic acid suppresses ultraviolet B eye irradiation-induced melanocyte activation by decreasing the levels of prohormone convertase 2 and alpha-melanocyte-stimulating hormone.

Tranexamic acid (trans-4-aminomethylcyclohexanecarboxylic acid) is a medicinal amino acid used in skin whitening care. This study examined the effects of tranexamic acid on the melanocyte activation of the skin induced by an ultraviolet (UV) B eye irradiation. The eye or ear was locally exposed to UVB at a dose of 1.0 kJ/m(2) using a 20SE sunlamp after covering the remaining body surface with aluminum foil. UVB eye irradiation induced melanocyte activation of the skin, similar to that observed following UVB ear irradiation, which was suppressed by the administration of tranexamic acid treatment. The plasma α-melanocyte-stimulating hormone (α-MSH) content was increased by UVB irradiation of the eye; however, the increase in α-MSH was suppressed by tranexamic acid treatment. In addition, UVB eye irradiation induced the up-regulation of prohormone convertase (PC) 2 in the pituitary gland. Meanwhile, the increase in PC2 induced by UVB eye irradiation was suppressed by tranexamic acid treatment. These results clearly indicate that tranexamic acid decreases the expression of PC2, which cleavages from proopiomelanocortin to α-MSH in the pituitary gland, thereby suppressing melanocyte activation.

Expression and function of bile acid receptor TGR5 in glucagon secreting pancreatic α cells: hyperglycemia induced expression of PC1 and release of GLP‐1 in response to activation of TGR5 by bile acids (1108.6)

We have recently shown that TGR5, a novel G protein coupled receptor, expressed in the pancreatic β cells induces insulin secretion in response to bile acids. Secretion of glucagon from the pancreatic α cells and glucagon‐like peptide 1 (GLP‐1) from the intestinal enteroendocrine cells also regulate insulin secretion. Glucagon and GLP‐1 are processed from the proglucagon by the cell‐specific expression of proconvertase 2 (PC2) and PC1, respectively. AIM. To determine expression and function of TGR5 in pancreatic α cells in normal and hyperglycemia. RESULTS. mRNA and protein expression of TGR5 was demonstrated in α cell line αTC1‐6 and mouse islets. Addition of INT‐777, a selective TGR5 ligand or lithocholic acid (LCA) increased glucagon secretion at low glucose (3 mM) in a concentration dependent manner in αTC1‐6 cells and mouse islets. PC2, but not PC1 was expressed in control αTC1‐6 cells. Treatment of α cells or mice islets with 25 mM glucose for 7 days induced expression of PC1 leading to secretion of GLP‐1 in response to INT‐777 and LCA. Similar release of GLP‐1 was obtained from diabetic db/db mice islets. CONCLUSION. Pancreatic α cells express TGR5 and PC2, and release glucagon in response to bile acids. Hyperglycemia induces expression of PC1 and causes release of GLP‐1. The importance of GLP‐1 in the regulation of insulin secretion underscores the significance of TGR5 as a potential therapeutic target in diabetes.

Two novel mutations affecting the same splice site of PKD1 correlate with different phenotypes in ADPKD

Genetic heterogeneity is the main factor for significant variation in the course of autosomal dominant polycystic kidney disease (ADPKD). PKD1 patients have more severe renal outcomes compared with PKD2 patients. Co-inheritance of a mutation in both genes is associated with more severe phenotypes than that found with either mutation alone. However, the genotype–phenotype relationship is far from clear in ADPKD. Here, we observed two novel mutations, PKD1:c.12444G > A and PKD1:c.12444 + 1G > A, which alter the same splice donor site of intron 45, correlate with different renal outcomes. To explain the phenomenon, we analyzed the genic and allelic background of the patients, as well as the genetic modifiers, DKK3 and HNF-1β as suggested. Only PKD1 variants were found, which highlights the allelic influence of PKD1 gene to be the last candidate factor. Segregation analysis, online mutation prediction, and recurrence mutation searching were applied to sort the variants. However, none of variants was found to be damaging or associated with the disease except PKD1:c.12444G > A and PKD1:c.12444 + 1G > A. Cloning and sequencing of the mutated cDNA sequences had shown unexpected different splicing effects caused by the mutations. PKD1:c.12444 + 1G > A definitely destroyed the native splice site and created a novel donor site with truncating effect on PC1. In contrast, PKD1:c.12444G > A mainly weakened the site and decreased the expression of normal PC1. Since PC1 negatively regulates cell proliferation in the process of cyst formation and enlargement, our observation may explain this new genotype–phenotype correlation and help to improve genetic counseling and diagnosis of the disease.

Differential processing of neuropeptide proprotein in human breast adenocarcinoma.

The processing of proprotein convertase (PC)-mediated neuropeptide plays a very important role in carcinogenesis and tumor proliferation. To investigate proneuropeptide processing mechanism in tumorigenesis and tumor proliferation. The expression and processing profiles of PC1, carboxypeptidase E (CPE), PC2, GHRH, or neuropeptide Y (NPY) gene and protein level were investigated between 42 human breast tumor tissues and 21 tumor-adjacent normal tissues. Gene analyses indicated that the proPC1, CPE, or preproNPY gene had higher expression in the breast tumor tissues, whereas the proPC2 or preproGHRH gene showed lower expression in the tissues. Protein analyses showed that the proPC1, PC1, CPE, GHRH, and preproNPY proteins were upregulated in the tumor tissues, whereas the proPC2, PC2, preproGHRH, and NPY proteins were down-regulated in them. The tissue results were highly corroborated with the serum data from the tumor patients and healthy women. The higher PC1 and CPE expressions as well as the transformation of more proGHRH into active GHRH peptide suggest stronger PC1/CPE-mediated neuropeptide processing in the tumor, whereas the lower PC2 expression as well as the transformation of less proNPY into active NPY peptide suggests a weak PC2-mediated processing in it. The alterations of the convertase expressions and processing show that there is a differential proprotein processing system in the tumor, which leads to the abnormal distributions of species, ratio, and concentration of (pro)peptide(s) in the microenvironment of cells. The latter may contribute to cancer progression.

Ultraviolet B irradiation of the mouse eye induces pigmentation of the skin more strongly than does stress loading, by increasing the levels of prohormone convertase 2 and α-melanocyte-stimulating hormone

In previous studies, we made the unexpected finding that in mice, ultraviolet (UV)B irradiation of the eye increased the concentration of α-melanocyte-stimulating hormone (α-MSH) in plasma, and systemically stimulated epidermal melanocytes. To compare the extent of the pigmentation induced by social and restraint stress (which activate the hippocampus-pituitary system) with that induced by UVB irradiation. DBA/2 and sham-operated or hypophysectomized DBA/2 mice were subjected to local UVB exposure using a sunlamp directed at the eye, and two types of stress (social and restraint) were imposed. UVB irradiation of the eye or exposure to stress loading both increased the number of Dopa-positive melanocytes in the epidermis, and hypophysectomy strongly inhibited the UVB-induced and stress-induced stimulation of melanocytes. Irradiation of the eye caused a much greater increase in dopamine than did the stress load. Both UVB eye irradiation and stress increased the blood levels of α-MSH and adrenocorticotropic hormone (ACTH). In addition, the increase in plasma α-MSH was greater in animals subjected to UVB eye irradiation than in those subjected to stress loading, whereas the reverse occurred for plasma ACTH. UVB irradiation to the eye and stress loading increased the expression of prohormone convertase (PC)1/3 and PC2 in the pituitary gland. The increase in expression of pituitary PC2 was greater in animals subjected to UVB eye irradiation than to stress, whereas no difference was seen between the two groups for the increase in PC1/3. UVB eye irradiation exerts a stronger effect on pigmentation than stress loading, and is related to increased levels of α-MSH and PC2.

Mild exercise suppresses exacerbation of dermatitis by increasing cleavage of the β-endorphin from proopiomelanocortin in NC/Nga mice

This study investigated the mechanism by which the strength and weakness of exercise stress affects the skin symptoms of atopic dermatitis (AD). Specific pathogen-free (SPF) and conventional NC/Nga mice were used. Conventional mice, but not the SPF, spontaneously develop dermal symptoms similar to that of patients with AD. There were two types of stress, mild (20 m/min for 60 min) or strong exercise (25 m/min for 90 min), using a treadmill four times per day. The symptom of the conventional group were strongly exacerbated by strong exercise but ameliorated by mild exercise. The plasma concentrations of α-melanocyte stimulating hormone (α-MSH) and the expression of melanocortin receptor-1 in skin elevated after strong exercise but decreased after mild exercise. The plasma levels of β-endorphin and the expression of µ-opioid receptor in skin were increased by mild exercise. In addition, the expression of prohormone convertase (PC) 1/3, PC2 and carboxypeptidase E (CPE) in pituitary gland were higher in the conventional group than in the SPF group. The level of PC2 was suppressed by mild exercise in the conventional groups, and elevated further by strong exercise. The level of PC1/3 becomes higher with the increase of the exercise load. On the other hand, the expression of the CPE was further increase by mild exercise but suppressed by strong exercise. These observations suggested that exercise-induced stress significantly affect the symptoms of AD in a pivotal manner depending on the levels of α-MSH and β-endorphin, and the expression of pituitary PC2 and CPE.

236 - Polycystins 1 and 2 as Novel Prognostic Markers in Colorectal Cancer

ABSTRACT Background/purpose Polycystins 1 and 2 (PC-1 and PC-2) constitute a family of cellular proteins detected in a variety of epithelial cells throughout human tissues. They function as mechanosensors and contribute to cellular homeostasis through proliferation, differentiation and apoptotic processes. Nevertheless, their fundamental role in cell-to-cell mechanical interactions, cell–matrix communication, tissue morphogenesis and planar cell polarity connote their engagement in processes of oncogenesis, including invasion and metastasis. The aim of the study was to investigate the expression of PC-1 and PC-2 in colorectal cancer (CRC), identify possible correlations with histopathological characteristics and explore the underpinning signal transduction mechanisms. Methods The immunohistochemical expression of PC-1 and PC-2 was evaluated in paraffin sections of 144 CRC patients and the results were statistically correlated with clinical and pathologic parameters. Molecular mechanisms were explored by Western blot, immunofluorescence and flow cytometry in SW480 CRC cell line expressing PC-1 and PC-2. Results Significantly elevated cytoplasmic expression was observed for PC-1 and PC-2 in mucinous carcinomas (p = 0.0035 and p = 0.0001, respectively). High expression levels of PC-2 were correlated with advanced TNM stage (p = 0.0324) and depth of tumour invasion (p = 0.0311). High expression levels of PC-1 (p = 0.0078) and altered expression levels of PC-2 in the transition zone from normal to cancerous tissue (p = 0.0198) were associated with reduced five-year survival rate. Altered expression of PC-1 in the transition zone was correlated with higher chance of relapse for the patients (p = 0.0019). Experiments in SW480 cells revealed funneling of PC-1 and PC-2 function via mammalian target of rapamycin (mTOR) and p70S6 kinases. Conclusions Our findings suggest that PC-1 and PC-2 are implicated in the development of an aggressive phenotype in CRC, as well as in reduced five-year survival rate and cancer-free survival, thus constituting potential prognostic markers. Their function seems to be partially mediated by key molecules of the mTOR signaling pathway. Disclosure All authors have declared no conflicts of interest.

Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst development and enlargement, as well as cell growth, are associated with alterations in several pathways, including cAMP and activator protein 1 (AP1) signalling. However, the precise mechanism by which these molecules stimulate cell proliferation is not yet fully understood. We now show by microarray analysis, luciferase assay, mutagenesis, and chromatin immunoprecipitation that CREB and AP1 contribute to increased expression of the amphiregulin gene, which codifies for an epidermal growth factor-like peptide, in ADPKD cystic cells, thereby promoting their cell growth. Increased amphiregulin (AR) expression was associated with abnormal cell proliferation in both PKD1-depleted and -mutated epithelial cells, as well as primary cystic cell lines isolated from ADPKD kidney tissues. Consistently, normal AR expression and proliferation were re-established in cystic cells by the expression of a mouse full-length PC1. Finally, we show that anti-AR antibodies and inhibitors of AP1 are able to reduce cell proliferation in cystic cells by reducing AR expression and EGFR activity. AR can therefore be considered as one of the key activators of the growth of human ADPKD cystic cells and thus a new potential therapeutic target.

Upregulation of β-cell genes and improved function in rodent islets following chronic glucokinase activation

Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to β-cell exhaustion; this study examines the effect of chronic GK activation on β-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key β-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk(del/wt)). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPβ compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk(del/wt) islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key β-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.

Polycystin-1, 2, and STIM1 Interact with IP3R to Modulate ER Ca2+ Release through the PI3K/Akt Pathway

Dysregulation of Ca²⁺ signaling and homeostasis has been linked to the development of ADPKD through aberrant functioning of the polycystins. In this study, we investigated the role of the polycystins in modulating Ca²⁺ signaling. Expression of full-length PC1 in MDCK cells inhibited intracellular Ca²⁺ release in response to ATP when compared to control cells. This phenotype correlated with reduced interaction of endogenous PC2 and IP₃R in PC1-containing cells. We also found that endogenous STIM1 also interacted with the IP₃R, and this interaction was enhanced by PC1 expression. Increased interaction between STIM1 and IP₃R inhibited Ca²⁺ release. PC1 regulates intracellular Ca²⁺ release and the interaction of PC2-IP₃R-STIM1 through the PI3K/Akt signaling pathway. Inhibition of the PI3K/Akt pathway in PC1 containing cells restored intracellular Ca²⁺ release, increased the interaction between PC2 and IP₃R and disrupted the STIM1-IP₃R complex. Conversely, activation of the PI3K/Akt signaling pathway by HGF in control MDCK cells gave the reverse effects. It reduced the release of Ca²⁺ to levels comparable to the PC1 cells, inhibited the association PC2 and IP₃R, and increased the interaction between STIM and IP₃R. Overall, our studies provide a potential mechanism for the modulation of intracellular Ca²⁺ signaling by the polycystins.

PC-1 enhances &lt;I&gt;c-myc&lt;/I&gt; gene expression in prostate cancer cells

PC-1(Prostate and colon gene 1) gene belongs to TPD52 (Tumor Protein D52) gene family. The expression of PC-1 is found to promote androgen-independent progression. This study was conducted to assess the mechnism of promotion of androgen-independent progression in PC-1 gene. The c-myc gene expression was tested by RT-PCR and Western blotting analyses in the LNCaP-pc-1 and LNCaP-zero cell line. After separation of cytoplasm and nulear proteins of the LNCaP-pc-1 and LNCaP-zero cell line, the beta-catenin protein was detected by Western blotting. C4-2 cell line was used to examine the effects of 10058-F4 on the PC-1 gene expression. The results of RT-PCR and Western blotting indicated that PC-1 enhanced c-myc gene expression in prostate cancer cells, PC-1 was also found to enhance beta-catenin expression in nuclear. Furthermore, a small-molecule c-Myc inhibitor, 10058-F4 represses PC-1 gene expression in C4-2 cell line. Our findings suggest that PC-1 enhances c-myc gene expression in prostate cancer cells through the Wnt/beta-catenin pathway. Meanwhile, c-myc plays a feed-forward role in enhancing PC-1 driven c-myc gene expression, and promotes prostate an-drogen-independent progression.

Abstract 2968: Aberrant expression of polycystin-1 in renal cell tumors

Abstract Polycystin-1 (PC1) is a cellular transmembrane protein encoded by the polycystic kidney disease gene (PKD1) prevalently expressed in developing/mature kidney and in autosomal polycystic kidney disease (ADPKD). Limited data are available concerning the PC1 involvement in renal tumorigenesis. Polycystin-1 expression was evaluated by immunohistochemistry (IHC) in 8 clear cell renal cell carcinomas (RCCs), 7 tubulopapilary cell type tumors, 3 solid RCCs developed in patients with von Hippel-Lindau disease (VHL), one RCC developed in a patient on chronic haemodyalisis and one angyomyolipoma in a patient with Tuberous sclerosis (TS). In parallel, the BIZ cell line derived from a high grade clear cell RCC and the WUR cell line derived from a tubulopapillary type RCC, were used to measure the level of PC1 expression by both immunolabeling and immunoblotting analyses. Five cases of clear cell type RCCs, and two tubulopapillary cell type RCCs consistently expressed PC1 with a percentage of labeled neoplastic cells ranging between 15% and 65%. Un the VHL disease-associated renal cell carcinoma, both the tumor cells and cystic tissues areas weakly expressed the PC1 protein. In TS-associated angiomyolipoma, the vascular component was PC1 positive, while the tumor cells were scarcely stained. High level of expression of PC1 was confirmed by both IHC and immunoblotting in WUR RCC cells (80% of labeled cells), while BIZ RCC cells exhibited a moderate PC1 staining (30% of labeled cells). Comparatively, in the normal kidney, consistent level of polycystin 1 was detected in distal tubules, collecting duct, glomerular podocytes and vascular smooth muscle cells. The strongest immunoreactivity against PC1 was observed in epithelial cells lining the cystic components in all ADPKD tissues. In conclusion, the presented data indicate consistent level of expression of the polycystin-1 protein in a number renal cell carcinomas and suggest that PC1 expression is linked to the tumor cell type, being more frequent event in clear cell type RCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2968.