The proprotein convertase PC5/6 is protective against intestinal tumorigenesis: in vivo mouse model

BACKGROUND: The secretory basic amino acid-specific proprotein convertases (PCs) have often been associated with cancer/metastasis. By controlling the cleavage of cancer-associated proteins, PCs play key roles in multiple steps of cancer development. Most analyses of the implication of PCs in cancer/metastasis relied on the use of in vitro overexpression systems or inhibitors that can affect more than one PC. Aside from the role of furin in salivary gland tumorigenesis, no other in vivo genetic model of PC-knockout was reported in relation to cancer development. RESULTS: Since PC5/6 is highly expressed in the small intestine, the present study examined its in vivo role in intestinal tumorigenesis. Analysis of human intestinal tumors at various stages showed a systematic down-regulation of PC5/6 expression. Since gene inactivation of PC5/6 leads to lethality at birth, we generated mice lacking PC5/6 in enterocytes and analyzed the impact of the presence or absence of this PC in the mouse ApcMin/+ model that develops numerous adenocarcinomas along the intestinal tract. This resulted in viable mice with almost no expression of PC5/6 in small intestine, but with no overt phenotype. The data showed that by themselves ApcMin/+ tumors express lower levels of PC5/6 mRNA, and that the lack of PC5/6 in enterocytes results in a significantly higher tumor number in the duodenum, with a similar trend in other intestinal segments. Finally, the absence of PC5/6 is also associated with a premature mortality of ApcMin/+ mice. CONCLUSION: Overall, these data suggest that intestinal PC5/6 is protective towards tumorigenesis, especially in mouse duodenum, and possibly in human colon.This work was supported by Canadian Institutes of Health Research grant # 44363, a Canada Chair # 201652, and a Strauss foundation grant

Proteolytic processing of human prorenin in renal and non-renal tissues

Proteolytic processing of human prorenin in renal and non-renal tissues. Previous studies have demonstrated that the mouse proprotein convertase PC1 (mPC1) accurately cleaves human prorenin to generate active renin and that this processing event appears to require co-packaging in secretory granules. In the current study, we have tested human PC1 (hPC1; also called PC3) for its ability to activate human prorenin. Our results suggest that while hPC1 is capable of carrying out the specific cleavage of human prorenin, it does so at a reduced efficiency as compared to mPC1. This difference is due to sequences in the carboxy-terminus of PC1 as demonstrated by the activity of hybrid hPC1/mPC1 molecules. These studies demonstrate that PC1 cleavage of prorenin can occur in humans and identify a functionally important region in the hPC1 protein for this interaction. Moreover, the localization of PC1 in human tissues suggests that it may participate in the generation of active renin in the adrenal medulla and possibly in certain adrenal tumors

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

International audienceHuman coronaviruses (HCoV) are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S) glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43). In an attempt to study the role of this protein in virus spread within the central nervous system (CNS) and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758), which introduces a putative furin-like cleavage (↓) site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies

Regional processing of the N- and C-terminal domains of proopiomelanocortin in monkey pituitary and brain

The total content and extent of processing of the [gamma]3MSH and [beta]-endorphin-containing N- and C-terminal domains of proopiomelanocortin were determined in the anterior and intermediate lobes of the pituitaries and in 11 regions of the brains of three Rhesus monkeys. Most immunoreactive [gamma]3MSH and [beta]-endorphin was located in the pituitary lobes, although significant amounts were also found in several brain regions. Sephadex column chromatography revealed that [gamma]3MSH immunoreactivity was found primarily as 4K and 9K forms; no [gamma]1MSH was detected. [beta]-Endorphin immunoreactivity was found as [beta]-endorphin, [beta]-lipotropin, and as a 5K form which may represent [beta]-endorphin extended N-terminally by part or all of [beta]-MSH. In the anterior lobe of the pituitary, the predominant products were 9K [gamma]3MSH and [beta]-lipotropin; in the intermediate lobe, more processed forms (4K [gamma]3MSH, [beta]-endorphin and 5K [beta]-endorphin) appeared to be preferentially stored. The pattern of processing in various brain regions was similar to that of the intermediate lobe of the pituitary.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27343/1/0000368.pd

Abnormal expression and processing of the proprotein convertases PC1 and PC2 in human colorectal liver metastases

BACKGROUND: The family of proprotein convertases has been recently implicated in tumorigenesis and metastasis in animal models. However, these studies have not yet been completely corroborated in human tumors. METHODS: Using RT PCR, immunoblot and immunohistochemistry we assessed the presence and the processing patterns of the convertases PC1 and PC2 as well as the PC2 specific chaperone 7B2 in human liver metastases originating from colorectal cancer and compared them to unaffected and normal liver. Furthermore, we assessed the presence and processing profiles of PC1, PC2 and 7B2 in primary colon cancers. RESULTS: mRNA, protein expression, and protein cleavage profiles of proprotein convertases 1 and 2 are altered in liver colorectal metastasis, compared to unaffected and normal liver. Active PC1 protein is overexpressed in tumor, correlating with its mRNA profile. Moreover, the enhanced PC2 processing pattern in tumor correlates with the overexpression of its specific binding protein 7B2. These results were corroborated by immunohistochemistry. The specific and uniform convertase pattern observed in the metastases was present only in a fraction of primary colon cancers. CONCLUSION: The uniformly altered proprotein convertase profile in liver metastases is observed only in a fraction of primary colon cancers, suggesting possible selection processes involving PCs during metastasis as well as an active role of PCs in liver metastasis. In addition, the exclusive presence of 7B2 in metastatic tumors may represent a new target for early diagnosis, prognosis and/or treatment

Immunocytochemistry of the C-terminal peptide of propressophysin (CPP): Relationship to vasopressin, oxytocin and neurophysin

Arginine-vasopressin (AVP) and its associated neurophysin (AVP-NP) are synthesized via a precursor, propressophysin, which also contains a 39 amino acid glycopeptide at its C-terminus (C-terminus of propressophysin, or CPP). In the present study, immunocytochemical techniques were used to determine the cellular co-localization of CPP with AVP, oxytocin (OXY), AVP-NP and OXY-NP in the rat hypothalamus using colchicine pre-treatment and serial 5 [mu]m section analysis. Extensive cross-competition studies of antisera raised against each peptide with the various antigens yielded no significant crossreactivity of the CPP, AVP, OXY and NP antisera. The NP antiserum, although directed against both AVP-NP and OXY-NP, demonstrated a preference for OXY-NP at a dilution of 1:20,000. CPP and AVP were always co-localized within the same magnocellular neurons of the supraoptic, paraventricular and circularis nuclei, and further showed very similar patterning in the suprachiasmatic nucleus as well. In conrast, no cellular overlap could be detected between CPP and OXY, in any of the above nuclei (the suprachiasmatic nucleus is devoid of OXY). Likewise, no examples of co-localization of CPP and OXY-NP were found in the magnocellular nuclei. These results are in strong agreement with a biosynthetic relationship between CPP, AVP and AVP-NP, and their separateness from the OXY and OXY-NP precursor.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25176/1/0000615.pd

A Locked Nucleic Acid Antisense Oligonucleotide (LNA) Silences PCSK9 and Enhances LDLR Expression In Vitro and In Vivo

The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity.LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome

Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice

PC7 belongs to the proprotein convertase family, whose members are implicated in the cleavage of secretory precursors. The in vivo function of PC7 is unknown. Herein, we find that the precursor proBDNF is processed into mature BDNF in COS-1 cells coexpressing proBDNF with either PC7 or Furin. Conversely, the processing of proBDNF into BDNF is markedly reduced in the absence of either Furin or PC7 in mouse primary hepatocytes. In vivo we observe that BDNF and PC7 mRNAs are colocalized in mouse hippocampus and amygdala and that mature BDNF protein levels are reduced in these brain areas in PC7 KO mice but not in the hippocampus of PC1/3 KO mice. Various behavioral tests reveal that in PC7 KO mice spatial memory is intact and plasticity of responding is mildly abnormal. Episodic and emotional memories are severely impaired, but both are rescued with the tyrosine receptor kinase B agonist 7,8-dihydroxyflavone. Altogether, these results support an in vivo role for PC7 in the regulation of certain types of cognitive performance, in part via proBDNF processing. Because polymorphic variants of human PC7 are being characterized, it will be important in future studies to determine their effects on additional physiological and behavioral processes