PC2 Expression Research Articles

Weight-drop model as a valuable tool to study potential neurobiological processes underlying behavioral and cognitive changes secondary to mild traumatic brain injury

The pathophysiology of post-traumatic brain injury (TBI) behavioral and cognitive changes is not fully understood, especially in its mild presentation. We designed a weight drop TBI model in mice to investigate the role of neuroinflammation in behavioral and cognitive sequelae following mild TBI. C57BL/6 mice displayed depressive-like behavior at 72 h after mild TBI compared with controls, as indicated by a decrease in the latency to first immobility and climbing time in the forced swim test. Additionally, anxiety-like behavior and hippocampal-associated spatial learning and memory impairment were found in the elevated plus maze and in the Barnes maze, respectively. Levels of a set of inflammatory mediators and neurotrophic factors were analyzed at 6 h, 24 h, 72 h, and 30 days after injury in ipsilateral and contralateral hemispheres of the prefrontal cortex and hippocampus. Principal components analysis revealed two principal components (PC), which represented 59.1% of data variability. PC1 (cytokines and chemokines) expression varied between both hemispheres, while PC2 (neurotrophic factors) expression varied only across the investigated brain areas. Our model reproduces mild TBI-associated clinical signs and pathological features and might be a valuable tool to broaden the knowledge regarding mild TBI pathophysiology as well as to test potential therapeutic targets.

#3679 ANOCTAMIN 1 INHIBITION SUPPRESSES CYSTOGENESIS BY ENHANCING CILIOGENESIS AND THE CILIARY DOSAGE OF POLYCYSTINS

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is a ciliopathy which is characterized by abnormal tubular epithelial proliferation and fluid secretion. Anoctamin 1 (ANO1) is a calcium-dependent chloride channel, which has recently been shown to be involved in ADPKD progression. However, how ANO1 contributes to ADPKD is largely unexplored. Method 1. Based on mRNA gene chips, the expression of ANO1 abnormally increased in human ADPKD kidneys. 2. Immunohistochemical staining showed ANO1 location. 3. Real-time PCR and Western blot showed mRNAs and protein of ANO1 in ADPKD patients and animal model-PKD1RC/RC mice. 4. Confocal immunofluorescence showed ANO1 and PC2 location in the cilia. 5. 3D gel culture to verify the function of ANO1 in cysts formation. 6. ANO1 inhibitors and siRNA to discover STAT3 and ERK pathway in ADPKD cell by WB. Results We found that ANO1 is consistently upregulated inhuman and mouse PKD kidneys. Intriguingly, ANO1 located in a vesicle-like patten at the ciliary base but not on the ciliary surface. ANO1 deficiency promotes both ciliogenesis and the ciliary dosage of polycystin-2 in human Pkd1-/- cells, and reduced cyst formation in 3D culture models. Moreover, inhibition of ANO1 abolished the activation of STAT3 and ERK pathways in PKD cells. Conclusion Our data indicate ANO1 may aggravate ADPKD by regulating the cilia length and PC2 expression. Moreover, ANO1 could support cyst growth by activating STAT3 and ERK pathways. Thus ANO1 is a negative regulator for both cilia formation and cilia trafficking of polycystins and provide mechanistic insights regarding the therapeutic potential of ANO1 pathway in ADPKD treatment.

Cell-specific expression and individual function of prohormone convertase PC1/3 in Tribolium larval growth highlights major evolutionary changes between beetle and fly neuroendocrine systems

BackgroundThe insect neuroendocrine system acts in the regulation of physiology, development and growth. Molecular evolution of this system hence has the potential to allow for major biological differences between insect groups. Two prohormone convertases, PC1/3 and PC2, are found in animals and both function in the processing of neuropeptide precursors in the vertebrate neurosecretory pathway. Whereas PC2-function is conserved between the fly Drosophila and vertebrates, ancestral PC1/3 was lost in the fly lineage and has not been functionally studied in any protostome.ResultsIn order to understand its original functions and the changes accompanying the gene loss in the fly, we investigated PC1/3 and PC2 expression and function in the beetle Tribolium castaneum. We found that PC2 is broadly expressed in the nervous system, whereas surprisingly, PC1/3 expression is restricted to specific cell groups in the posterior brain and suboesophageal ganglion. Both proteases have parallel but non-redundant functions in adult beetles’ viability and fertility. Female infertility following RNAi is caused by a failure to deposit sufficient yolk to the developing oocytes. Larval RNAi against PC2 produced moulting defects where the larvae were not able to shed their old cuticle. This ecdysis phenotype was also observed in a small subset of PC1/3 knockdown larvae and was strongest in a double knockdown. Unexpectedly, most PC1/3-RNAi larvae showed strongly reduced growth, but went through larval moults despite minimal to zero weight gain.ConclusionsThe cell type-specific expression of PC1/3 and its essential requirement for larval growth highlight the important role of this gene within the insect neuroendocrine system. Genomic conservation in most insect groups suggests that it has a comparable individual function in other insects as well, which has been replaced by alternative mechanisms in flies.

Principal components of tau positron emission tomography and longitudinal tau accumulation in Alzheimer\u2019s disease

BackgroundWe aimed to investigate the clinical correlates of principal components (PCs) of tau positron emission tomography (PET) and their predictability for longitudinal changes in tau accumulation in Alzheimer’s disease (AD).MethodsWe enrolled 272 participants who underwent two PET scans [18F-flortaucipir for tau and 18F-florbetaben for amyloid-β (Aβ)], brain magnetic resonance imaging, and neuropsychological tests as baseline assessments. Among them, 187 participants underwent the same follow-up assessments after an average of 2 years. Using Aβ-positive AD dementia-specific PCs obtained from the baseline scans of 56 Aβ-positive patients with AD dementia, we determined the expression of the first two PCs (PC1 and PC2) in all participants. We assessed the correlation of PC expression with baseline clinical characteristics and tau accumulation rates. Moreover, we investigated the predictability of PCs for the longitudinal tau accumulation in training and test sets.ResultsPC1 corresponded to the tau distribution pattern in AD, while the two PC2 extremes reflected the parietal or temporal predominance of tau distribution. PC1 expression increased with tau burden and decreased with cognitive impairment, while PC2 expression decreased with advanced age and visuospatial and attention function deterioration. The tau accumulation rate was positively correlated with PC1 expression (greater tau burden) and negatively correlated with PC2 expression (temporal predominance). A regression model using both PCs could predict longitudinal changes in the tau burden (intraclass correlation coefficient = 0.775, R2 = 0.456 in test set).ConclusionsPC analysis of tau PET could be useful for evaluating disease progression, characterizing the tau distribution pattern, and predicting longitudinal tau accumulation.

Polycystin 2 is increased in disease to protect against stress-induced cell death

Polycystin 2 (PC2 or TRPP1, formerly TRPP2) is a calcium-permeant Transient Receptor Potential (TRP) cation channel expressed primarily on the endoplasmic reticulum (ER) membrane and primary cilia of all cell and tissue types. Despite its ubiquitous expression throughout the body, studies of PC2 have focused primarily on its role in the kidney, as mutations in PC2 lead to the development of autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition for which there is no cure. However, the endogenous role that PC2 plays in the regulation of general cellular homeostasis remains unclear. In this study, we measure how PC2 expression changes in different pathological states, determine that its abundance is increased under conditions of cellular stress in multiple tissues including human disease, and conclude that PC2-deficient cells have increased susceptibility to cell death induced by stress. Our results offer new insight into the normal function of PC2 as a ubiquitous stress-sensitive protein whose expression is up-regulated in response to cell stress to protect against pathological cell death in multiple diseases.

Profiles of pro-opiomelanocortin and encoded peptides, and their processing enzymes in equine pituitary pars intermedia dysfunction.

Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study was that PC1 and PC2 expression in the pituitary gland are altered in PPID, resulting in an abnormal relative abundance of POMC derived proteins. The objectives of this study were to identify the partial sequences of equine POMC, PC1, and PC2 mRNAs; and to determine whether the expression of POMC, PC1, and PC2 mRNAs in whole pituitary extracts, and POMC-protein in the cavernous sinus blood of horses are altered in PPID. We confirmed (RT-PCR and sequencing) that the partial sequences obtained match the corresponding regions of predicted equine POMC, PC1 and PC2 sequences. The expression (quantification by RT-qPCR) of POMC, PC1 and PC2 mRNAs were found upregulated in the pituitary of horses with PPID. Plasma (measured using RIA/ELISA) ACTH and α-MSH were elevated in PPID horses. These results indicate distinct differences in gene and protein expression of POMC and its intermediates, and processing enzymes in PPID. It provides evidence to support the notion that local, pituitary-specific inadequacies in prohormone processing likely contribute to equine PPID.

Metabolic regulation of GLP-1 and PC1/3 in pancreatic α-cell line.

Background and aimsAn intra-islet incretin system has been recently suggested to operate through modulation of the expression and activity of proconvertase 1/3 and 2 (PC1/3, PC2) in pancreatic alpha-cell accounting for local release of GLP-1. Little is known, whether this alpha-cell activity can be affected by the metabolic alterations occurring in type 2 diabetes, such as hyperglycemia, hyperlipidemia or hyperglucagonemia.Materials and methodsAlphaTC1/6 cells from a mice pancreatic cell line were incubated in the presence of two glucose (G) concentration (5.5 and 16.7 mM) for 16 h with or without free fatty acid, IL6 or glucagon. GLP-1 secretion was measured by ELISA and expression of PC1/3 and PC2 by RT-PCR and western blot; cell viability was determined by MTT method, Reactive Oxygen Species generation (ROS) by H2DCFDA fluorescence and apoptosis by Annexin staining and Propidium Iodine (PI) fluorescence.ResultsUpon 16.7G incubation, GLP-1 secretion (total and active) was significantly increased in parallel with a significant increment in PC1/3 expression, a slight increase in cell viability and ROS generation and by a decrement in PC2 expression with no change in cell apoptosis. When cells were incubated at 5.5mM glucose with FFA, also an increment in GLP-1 secretion and PC1/3 expression was observed together an increment in ROS generation, a decrement in cell viability, and a modest increment in apoptosis. When incubated with 16.7mM glucose with FFA, the increment in GLP-1 secretion was reduced to basal, accompanied by an increment in apoptosis and ROS generation. This was also observed with IL-6, but in this case, no modification in ROS generation or apoptosis was observed when compared to 16.7mM glucose. The presence of glucagon did not modify any of the parameters studied.ConclusionThese data suggest that under hyperglycemic, hyperlipidemia or inflammatory conditions, alpha cells can increase expression PC1/3 and activate GLP-1 secretion, which may contribute protecting both alpha and beta-cells from glucose and lipotoxicity, while this effect seems to be lost in the presence of both pathophysiological conditions.

External Ca2+ regulates polycystin-2 (TRPP2) cation currents in LLC-PK1 renal epithelial cells

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel whose dysfunction is associated with the onset of autosomal dominant polycystic kidney disease (ADPKD). PC2 contributes to Ca2+ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to the external Ca2+ regulation of PC2 channel function. In this study, we explored the effect of external Ca2+ on endogenous PC2 in wild type LLC-PK1 renal epithelial cells. We obtained whole cell currents at different external Ca2+ concentrations, and observed that the basal whole cell conductance in normal Ca2+(1.2mM), decreased by 30.2% in zero (nominal) Ca2+ and conversely, increased by 38% in high external Ca2+(6.2mM). The high Ca2+-increased whole cell currents were completely inhibited by either PC2 gene silencing, or intracellular dialysis with active, but not denatured by boiling, PC2 antibody. Exposure of cells to high Ca2+ was also associated with relocation of PC2 to the plasma membrane. To explore whether a Ca2+ sensing receptor (CaSR) was implicated in the external Ca2+ modulation of PC2 currents, we tested the effect of the CaSR agonists, spermine and the calcimimetic R-568, which largely mimicked the effect of high Ca2+ under Ca2+-free conditions. The CaSR agonist gentamicin also increased the PC2 currents in the presence of normal Ca2+. The presence of CaSR was confirmed by immunocytochemistry, which partially colocalized with the intracellular PC2 protein, in an external Ca2+-dependent manner. The data support a novel Ca2+ sensing mechanism for PC2 expression and functional regulation in renal epithelial cells.

Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation

Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.

Acute kidney injury induces hallmarks of polycystic kidney disease.

Acute kidney injury (AKI) and autosomal dominant polycystic kidney disease (ADPKD) are considered separate entities that both frequently cause renal failure. Since ADPKD appears to depend on a polycystin-1 (Pc1) or Pc2 dosage mechanism, we investigated whether slow progression of cystogenesis in two Pkd1 transgenic mouse models can be accelerated with moderate ischemia-reperfusion injury (IRI). Transient unilateral left ischemic kidneys in both nontransgenic and transgenic mice reproducibly develop tubular dilatations, cysts, and typical PKD cellular defects within 3 mo post-IRI. Similar onset and severity of IRI induced-cystogenesis independently of genotype revealed that IRI is sufficient to promote renal cyst formation; however, this response was not further amplified by the transgene in Pkd1 mouse models. The IRI nontransgenic and transgenic kidneys showed from 16 days post-IRI strikingly increased and sustained Pkd1/Pc1 (>3-fold) and Pc2 (>8-fold) expression that can individually be cystogenic in mice. In parallel, long-term and important stimulation of hypoxia-inducible factor 1α expression was induced as in polycystic kidney disease. While mammalian target of rapamycin signaling is activated, stimulation of the Wnt pathway, with markedly increased active β-catenin and c-Myc expression in IRI renal epithelium, uncovered a similar regulatory cystogenic response shared by IRI and ADPKD. Our study demonstrates that long-term AKI induces cystogenesis and cross talk with ADPKD Pc1/Pc2 pathogenic signaling.

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.

Elevated expression of mechanosensory polycystins in human carotid atherosclerotic plaques: association with p53 activation and disease severity.

Atherosclerotic plaque formation is associated with irregular distribution of wall shear stress (WSS) that modulates endothelial function and integrity. Polycystins (PC)-1/-2 constitute a flow-sensing protein complex in endothelial cells, able to respond to WSS and induce cell-proliferation changes leading to atherosclerosis. An endothelial cell-culture system of measurable WSS was established to detect alterations in PCs expression under conditions of low- and high-oscillatory shear stress in vitro. PCs expression and p53 activation as a regulator of cell proliferation were further evaluated in vivo and in 69 advanced human carotid atherosclerotic plaques (AAPs). Increased PC-1/PC-2 expression was observed at 30–60 min of low shear stress (LSS) in endothelial cells. Elevated PC-1 expression at LSS was followed by p53 potentiation. PCs immunoreactivity localizes in areas with macrophage infiltration and neovascularization. PC-1 mRNA and protein levels were significantly higher than PC-2 in stable fibroatherotic (V) and unstable/complicated (VI) AAPs. Elevated PC-1 immunostaining was detected in AAPs from patients with diabetes mellitus, dyslipidemia, hypertension and carotid stenosis, at both arteries (50%) or in one artery (90%). PCs seem to participate in plaque formation and progression. Since PC-1 upregulation coincides with p38 and p53 activation, a potential interplay of these molecules in atherosclerosis induction is posed.

Regulation of prohormone convertase 2 protein expression via GPR40/FFA1 in the hypothalamus

Previous studies have shown that the administration of docosahexaenoic acid (DHA) or GW9508, a GPR40/FFA1 (free fatty acid receptor) agonist, facilitates β-endorphin release in the arcuate nucleus of the hypothalamus in mice. However, the mechanisms mediating β-endorphin release induced by GPR40/FFA1 agonists remain unknown. In this study, we focused on the changes in expression of hypothalamic prohormone convertase (PC) 2, which is a calcium-dependent subtilisin-related proteolytic enzyme. The intracerebroventricular injection of DHA or GW9508 significantly increased PC2 protein expression in the hypothalamus. This increase in PC2 expression was inhibited by pretreatment with GW1100, a GPR40/FFA1 antagonist. Furthermore, PC2 protein expression gradually increased over time after complete Freund's adjuvant. These increase in PC2 expression were inhibited by pretreatment with GW1100. However, GW1100 by itself had no effect on PC2 levels. Taken together, our findings suggest that activation of the hypothalamic GPR40/FFA1 signaling pathway may regulate β-endorphin release via PC2, and regulate the endogenous pain control system.

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.

Polycystin-1 and polycystin-2 are involved in the acquisition of aggressive phenotypes in colorectal cancer.

The polycystins PC1 and PC2 are emerging as major players in mechanotransduction, a process that influences all steps of the invasion/metastasis cascade. We hypothesized that PC1 and PC2 facilitate cancer aggressiveness. Immunoblotting, RT-PCR, semi-quantitative and quantitative real-time PCR and FACS analyses were employed to investigate the effect of polycystin overexpression in colorectal cancer (CRC) cells. The impact of PC1 inhibition on cancer-cell proliferation was evaluated through an MTT assay. In vitro data were analyzed by Student's t-test. HT29 human xenografts were treated with anti-PC1 (extracellular domain) inhibitory antibody and analyzed via immunohistochemistry to determine the in vivo role of PC1 in CRC. Clinical significance was assessed by examining PC1 and PC2 protein expression in CRC patients (immunohistochemistry). In vivo and clinical data were analyzed by non-parametric tests, Kaplan-Meier curves, log-rank test and Cox model. All statistical tests were two-sided. PC1 overexpression promotes epithelial-to-mesenchymal transition (EMT) in HCT116 cells, while PC2 overexpression results in upregulation of the mTOR pathway in SW480 cells. PC1 inhibition causes reduced cell proliferation in CRC cells inducing tumor necrosis and suppressing EMT in HT29 tumor xenografts. In clinical study, PC1 and PC2 overexpression associates with adverse pathological parameters, including invasiveness and mucinous carcinomas. Moreover, PC1 overexpression appears as an independent prognostic factor of reduced recurrence-free survival (HR = 1.016, p = 0.03) and lowers overall survival probability, while aberrant PC2 expression predicts poor overall survival (p = 0.0468). These results support, for the first time, a direct link between mechanosensing polycystins (PC1 and PC2) and CRC progression.

GLP-1 analog liraglutide enhances proinsulin processing in pancreatic β-cells via a PKA-dependent pathway.

Hyperproinsulinemia has gained increasing attention in the development of type 2 diabetes. Clinical studies have demonstrated that glucagon-like peptide-1 (GLP-1)-based therapies significantly decrease plasma proinsulin/insulin ratio in patients with type 2 diabetes. However, the underlying mechanism remains unclear. Prohormone convertase (PC)-1/3 and PC2 are primarily responsible for processing proinsulin to insulin in pancreatic β-cells. We have recently reported that Pax6 mutation down-regulated PC1/3 and PC2 expression, resulting in defective proinsulin processing in Pax6 heterozygous mutant (Pax6(m/+)) mice. In this study, we investigated whether and how liraglutide, a novel GLP-1 analog, modulated proinsulin processing. Our results showed that liraglutide significantly up-regulated PC1/3 expression and decreased the proinsulin to insulin ratio in both Pax6(m/+) and db/db diabetic mice. In the cultured mouse pancreatic β-cell line, Min6, liraglutide stimulated PC1/3 and PC2 expression and lowered the proinsulin to insulin ratio in a dose- and time-dependent manner. Moreover, the beneficial effects of liraglutide on PC1/3 and PC2 expression and proinsulin processing were dependent on the GLP-1 receptor-mediated cAMP/protein kinase A signaling pathway. The same mechanism was recapitulated in isolated mouse islets. In conclusion, liraglutide enhanced PC1/3- and PC2-dependent proinsulin processing in pancreatic β-cells through the activation of the GLP-1 receptor/cAMP/protein kinase A signaling pathway. Our study provides a new mechanism for improvement of pancreatic β-cell function by the GLP-1-based therapy.

Decreased Hypothalamic Prohormone Convertase Expression in Huntington Disease Patients

In Huntington disease (HD), hypothalamic neuropeptidergic systems are not equally affected at the peptide and mRNA levels. Because prohormone convertases (PCs) are critically involved in the conversion of propeptides into their active forms, we postulated that a decrease in PC expression may underlie these discrepancies. Therefore, we assessed the expression of PC1/3 and PC2 in the hypothalamic infundibular, suprachiasmatic, and paraventricular nuclei in postmortem tissues of HD patients and controls (n = 9, each) using immunocytochemistry and quantitative reverse transcription polymerase chain reaction. We also assessed PC1/3 and PC2 mRNA expression in the inferior frontal gyrus and colocalization of both PCs with corticotropin-releasing hormone and α-melanocyte-stimulating hormone. In HD patients, PC1/3 and PC2 expression was decreased in the hypothalamic infundibular (both p = 0.046) and paraventricular nuclei (p = 0.031 and p = 0.019). In the suprachiasmatic nucleus, PC1/3 and PC2 expressions were not different between HD and control cases; PC1/3 and PC2 mRNA levels in the inferior frontal gyrus were also not different. None of the PCs was colocalized with corticotropin-releasing hormone, whereas α-melanocyte-stimulating hormone showed colocalization with PC1/3 and PC2. These data suggest that defects in the processing of hypothalamic neuropeptides in HD may partially arise from decreased PC1/3 and PC2 expressions. These changes might contribute to selective neuropathology underlying various clinical manifestations and may provide novel therapeutic targets in HD patients.

Mutant PAX6 downregulates prohormone convertase 2 expression in mouse islets

Transcriptional factor paired box 6 (PAX6) is very important for the development of the eyes, central nervous system, and pancreas. PAX6 mutations are associated with a diabetic phenotype and abnormal glucose metabolism. Our previous study showed that PAX6 directly bound to and activated the prohormone convertase 1/3 (Pc1/3) gene promoter and subsequently regulated proinsulin processing. Prohormone convertase 2 (PC2) is the essential enzyme for pancreatic proinsulin processing. To study the regulation of PAX6 in Pc2 expression, we did research on the pancreas of Pax6 R266Stop mutant mice, where truncated mutations happened in the C-terminal of the PAX6 protein. Our studies showed that the mutant PAX6 protein was stable and regulated the activity of Pc2 promoter as shown by luciferase activity assays. We found that the wild-type PAX6 protein imparts a transcriptional effect, and the mutant PAX6 can also regulate the downstream molecules. The results provide new insights into the mechanism of truncated PAX6 in regulating the functions of the pancreas and endocrine system.

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.

Progressive development of polycystic kidney disease in the mouse model expressing Pkd1 extracellular domain

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by slow progression of multiple cysts in both kidneys that lead to renal insufficiency in mid-life or later. ADPKD is associated with mutations mainly in the PKD1 gene (encoding polycystin-1 or PC1) and less frequently in the PKD2 gene (encoding polycystin-2 or PC2). To mimic naturally occurring human PKD1 mutations and gain insight into the PC1 extracellular domain function, four transgenic mouse lines were established with exclusively the extracellular domain of the Pkd1 gene (Pkd1(extra)) under endogenous transcriptional regulation. Expression of the Pkd1(extra) transgene was 2- to 80-fold above endogenous levels. Strikingly, the Pc1(extra) protein was more abundant, proportionally to the endogenous levels. All four transgenic mouse lines consistently displayed progressive renal cystic phenotype. Consequently, these transgenic mice reproducibly developed renal functional alterations similar to human ADPKD with proteinuria, renal insufficiency, anemia and died of renal failure late in life. In precystic kidneys, the Pkd1(extra) transgene modulated Pc2 expression and thereby, uncovered a potential Pc1-mutant/Pc2 pathogenic crosstalk mechanism. Moreover, the pathophysiologic mechanism also implicates c-myc, a major modulator of cystogenesis. Altogether, the novel Pkd1(extra) mouse model is the first Pc1 extracellular mutant that reproduces human ADPKD clinical progression and physiopathology.