19 proprotein convertases (PCs) in degenerating/regenerating peripheral nerves
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Journal Article↗
Biomedical subjects
M Marcinkiewicz
Publications and source records attributed to M Marcinkiewicz.
The pro-protein convertase PC1 is induced in the transected sciatic nerve and is present in cultured Schwann cells: comparison with PC5, furin and PC7, implication in pro-BDNF processing.
Injury of peripheral nerves induces expression of several pro-protein convertases (PCs) involved in processing of precursor proteins into their diverse active end-products. In this study, the focus was on convertase PC1 which, although undetectable in control nerves, is strongly induced in injured nerves. High concentrations of PC1 mRNA of 9.0, 5.5, 3.0, 2.5 and 1.6 kb were observed on day 4 post-lesion in proximal and distal segments. By in situ hybridization PC1 mRNA was detected in most of endoneurial cells, which were further identified by immunocytochemistry as myelin 2', 3'-cyclic nucleotide 3'-phosphodiesterase containing Schwann cells. PC1 mRNA and protein were also present in cultured Schwann cells also containing convertases PC5, furin and PC7 as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Mostly unprocessed pro-NGF of 35 kDa and pro-BDNF of 35 kDa were found on Western blotting of Schwann cells. Expression of exogenous neurotrophins by infection with vaccinia virus vector showed that mouse pro-NGF and rat pro-BDNF are cleaved intracellularly on smaller forms of 13.5 kDa NGF and 14 kDa BDNF. Infection experiments demonstrated that Schwann cells contain active processing enzymes. In conclusion, this work provides in vivo evidence of the presence of several PCs in the injured rat sciatic nerve and ex vivo in cultured Schwann cells.
Animals↗
Precursor convertases: an evolutionary ancient, cell-specific, combinatorial mechanism yielding diverse bioactive peptides and proteins.
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Amino Acid Sequence↗
Pex mRNA is localized in developing mouse osteoblasts and odontoblasts.
Mutations in PEX, a phosphate-regulating gene with homology to endopeptidase on the X chromosome, were recently identified in patients with X-linked hypophosphatemia (XLH), an inherited disorder of phosphate homeostasis characterized by growth retardation and rachitic and osteomalacic bone disease. To understand the mechanism by which loss of PEX function elicits the mutant phenotype, a study of its mRNA localization and ontogenesis was undertaken. Using the reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) with polyA+ RNA purified from mouse testis, a 337-bp Pex cDNA fragment was generated and cloned in the pCRII plasmid. The cDNA was used to generate sense and anti-sense Pex riboprobes for in situ hybridization (ISH) and Northern analysis. To survey a large number of different tissues, sagittal sections of embryos and newborn mice were examined. ISH showed the presence of Pex mRNA in osteoblasts and odontoblasts. Pex gene expression was detectable on Day 15 of embryonic development, which coincides with the beginning of intercellular matrix deposition in bones. Finally, Northern analysis of total RNA from calvariae and teeth of 3-day-old and adult mice showed that the abundance of the 7-kb Pex transcript is decreased in adult bones and in nongrowing teeth. The present study demonstrates that Pex mRNA is expressed in bones and teeth and suggests that this putative endopeptidase plays an important role in the development of these tissues.
Aging↗
Changing patterns of renal retinal dehydrogenase expression parallel nephron development in the rat.
We have recently characterized a cytosolic aldehyde dehydrogenase from rat kidney that functions as a retinal dehydrogenase (RALDH) and have cloned the corresponding gene. RALDH catalyzes the oxidation of retinal to retinoic acid, which regulates cell growth and differentiation by activating retinoic acid receptors. In situ hybridization demonstrates that RALDH mRNA expression is prominent in kidney in 2-day-old rats, is detected in lung and in epithelia of several tissues, but is not found in liver tissue. Retinal dehydrogenase activity peaks in kidney at Day 2 after birth and decreases gradually until adulthood, correlating well with RALDH expression. Weaker activity is also detectable in lungs but not in liver. Notably, distribution patterns of RALDH in kidney tissues are dramatically altered during postnatal development (P). From P0 to P6, hybridization is essentially concentrated within the marginal nephrogenic zone of the cortex. Expression progresses to deeper cortical layers from P12 to P16 and is intense in the medulla at P42, and focal expression is still detectable in the cortex. Immunocytochemical localization of RALDH in neonatal kidney shows staining mostly in cortical zone convoluted tubules and in adult rat shows staining in segments of distal and proximal tubules. These data suggest an important role for RALDH in modulating retinoic acid levels in different cell types during rat kidney development. The changing patterns of RALDH expression mirror stages of nephron formation in the developing rat kidney, strongly suggesting a central role for RALDH and thus for retinoids in controlling kidney development.
Aging↗
Role of epidermal growth factor (EGF) in oesophageal mucosal integrity.
Oesophageal mucosa has well established protective mechanisms, which operate within pre-epithelial, epithelial and post-epithelial compartments. Since refluxed acid and pepsin always act from the luminal side of the mucosa, protective factors like EGF, operating as a part of pre-epithelial defence, are thought to be pivotal in the maintenance of the integrity of the oesophageal mucosa. The significant contribution of salivary EGF to the quality of the oesophageal mucosal barrier has been demonstrated in an experimental setting and in a clinical scenario. Patients with low salivary EGF levels are predisposed to severe oesophageal damage if they develop gastro-oesophageal reflux and are a high-risk group for development of Barrett's oesophagus. Not only the salivary glands but also the human oesophagus has a profound ability to elaborate and release EGF. Some changes in luminal release of EGF during oesophageal mucosal exposure to intraluminal damaging factors imply its role in the oesophageal protective mechanisms. To exert biological effects within the oesophageal mucosal compartment, EGF requires binding to the ligand-binding domain of its receptor. This process results in receptor dimerisation, autophosphorylation and activation of intracellular signal transduction pathways. EGF receptors are localised on the basolateral and luminal aspect of the mucosal cells playing an important role in fast regeneration of oesophageal epithelium through the high mitotic activity of its proliferative zone. An increase in the rate of salivary EGF secretion during masticatory stimulation suggests its potential therapeutic benefit in the treatment of patients with damaged oesophageal mucosa.
Barrett Esophagus↗
[The role of selected serologic markers in the diagnosis of autoimmune hepatitis].
Autoimmune hepatitis (AIH) is a progressive inflammatory liver disease characterized by specific duration of symptoms (above 6 months), 3- to 10-fold elevation in serum aminotransferase activities, 2-fold elevation in gamma-globulin, high titers (above 1:40) of circulating autoantibodies. Antinuclear antibodies (ANA), smooth muscle antibodies (SMA), and antibodies to liver/kidney microsome type 1 (anti-LKM1) are the commonly available markers of AIH. SMA and ANA lack disease specificity and they are present commonly in different liver diseases. Anti-LKM1 usually connote a type 2 form of AIH which involves children and responds to corticosteroid therapy. Other autoantibodies, such as those against actin (SMA-AA), the liver-cytosolic antigen (anti-LC1), the liver-specific membrane lipoprotein (anti-LPS), asialoglycoprotein receptor (anti-ASGPR), have greater disease specificity but less availability.
Adult↗
Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway.
We studied the extent of cellular inhibitory activity of alpha1-antitrypsin Portland (alpha1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha1-PDX in AtT20 cells. Results revealed that alpha1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of alpha1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha1-PDX cells demonstrated that alpha1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.
Animals↗
Role of platelet-activating factor in Chinese hamster ovary cell responses to cholera toxin.
Cholera toxin (CT)-induced intestinal secretion and Chinese hamster ovary cell (CHO) elongation involves cyclic adenosine monophosphate and protein synthesis-dependent prostaglandin formation. We previously reported inhibition of CT-induced intestinal secretion and CHO elongation by platelet-activating factor (PAF) receptor antagonists and secretion of PAF by human intestinal epithelial cells exposed to CT. Herein, we show that PAF is involved after cAMP and that PAF, like CT, mediates prostaglandin E2 synthesis in CHO cells. CT-induced CHO elongation was blocked by specific PAF receptor antagonists, BN52021 and SR27417. SR27417 blocked dibutyryl cAMP-induced CHO elongation, but did not alter CHO elongation caused by PGE2. Neither CT-stimulated cAMP accumulation nor PGE2 production was inhibited by SR27417. Both PGE2 and PAF caused significant CHO elongation, but the latter did not stimulate significant cAMP production. In addition, PAF, like CT and dibutyryl cAMP, stimulated significant PGE2 production. Finally, the protein synthesis inhibitor cycloheximide, which completely blocks the effect of CT on prostaglandin synthesis, also blocked that of PAF, suggesting that PAF also mediates protein synthesis-dependent prostaglandin formation. We conclude that PAF is involved in CHO cytoskeletal responses to CT after the accumulation of cAMP and, like CT, PAF stimulates protein synthesis-dependent prostaglandin accumulation.
Alprostadil↗
Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic nuclei: selective regulation of PC1 in corticotrophin-releasing hormone parvocellular neurons mediated by glucocorticoids.
The prohormone convertases (PCs) are processing enzymes that activate proproteins via cleavage at specific single or pairs of basic residues. The hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON) are primary sites of biosynthesis of several neuroendocrine hormone precursors, including provasopressin (pro-AVP), pro-oxytocin (pro-OT), and procorticotrophin-releasing hormone (pro-CRH), which require post-translational processing to yield active products. Using in situ hybridization, we observed PC1 and PC5 mRNAs in PVN and SON magnocellular neurons, while PC2 mRNA was observed in both magnocellular and parvocellular PVN neurons as well as magnocellular SON neurons. Similar to furin, PC7 mRNA was expressed throughout the PVN and SON, whereas PACE4 mRNA levels were undetectable. Both immunohistochemical and Western blot studies were performed to demonstrate the presence of PC proteins and forms in the PVN and SON. Using double-labeling in situ hybridization, we examined the cellular colocalization of each PC mRNA with pro-AVP, pro-OT, and pro-CRH mRNAs in PVN and SON. PC1 mRNA was colocalized with both AVP and OT mRNA in PVN and SON magnocellular neurons. All AVP, OT, and CRH neurons expressed PC2. In contrast, PC5 mRNA was colocalized only with OT mRNA. We examined the effects of adrenalectomy (ADX) on PVN PC mRNA levels. PC1 mRNA levels were increased selectively within CRH/AVP parvocellular neurons but were unchanged in PVN magnocellular AVP or OT neurons. These results established the anatomical organization of each convertase and proneuropeptide substrates in the PVN and SON and suggested potential roles for each enzyme under resting and stimulated conditions.
Adrenalectomy↗
Pilocarpine-induced seizures are accompanied by a transient elevation in the messenger RNA expression of the prohormone convertase PC1 in rat hippocampus: comparison with nerve growth factor and brain-derived neurotrophic factor expression.
Several prohormone convertases that are involved in the posttranslational processing of precursor proteins, including neuropetides, hormones and neurotrophic factors, are produced in the central nervous system. These include enzymes named furin, PC1, PC2, PC5 and PACE4. To understand better the potential role played by prohormone convertases in the central nervous system we studied the expression of their messenger RNAs in the hippocampus of rats with pilocarpine-induced seizures. Moreover, we compared their expression patterns with those of neurotrophins such as nerve growth factor and brain-derived neurotrophic factor, which are up-regulated in the hippocampus during seizures. Pilocarpine (380 mg/kg, i.p.) induced seizure activity that appeared within the first hour and persisted for approximately 8 h. In situ hybridization showed transient increases in messenger RNA for nerve growth factor and brain-derived neurotrophic factor that peaked at 120 min in the hippocampus. Among the convertases studied, only PC1 messenger RNA displayed up-regulation, with temporal and topographic features comparable to those of nerve growth factor and brain-derived neurotrophic factor messenger RNA. The expression of furin, PC2 and PC5 messenger RNA changed little, while PACE4 was not expressed at all, both before and after pilocarpine administration. The highest increase in PC1 messenger RNA expression was found in granule cells of the dentate gyrus and, to a lesser extent, in the pyramidal layer of CA1 and CA3 subfields. Thus, in the rat hippocampus, the epileptiform activity induced by pilocarpine mediates a co-ordinated expression of messenger RNAs for PC1, nerve growth factor and brain-derived neurotrophic factor. Our findings suggest the involvement of PC1 in the processing of precursor proteins during seizure activity.
Animals↗
Esophagoprotective potential of cisapride. An additional benefit for gastroesophageal reflux disease.
Cisapride is a novel prokinetic agent that releases acetylcholine at the level of the myenteric plexus. Acetylcholine also plays a role in the secretory function of salivary glands evoked by intraesophagal mechanical and chemical stimulation, mediated through the esophagosalivary reflex. The impact, however, of cisapride on salivary protective components mediated by esophagosalivary reflex remains unknown. Therefore, we have studied salivary pH, bicarbonate, nonbicarbonate, glycoconjugate, protein, EGF, TGF-alpha, and PGE2 before and after the administration of cisapride. The study was conducted in 20 asymptomatic volunteers (9 women and 11 men, mean age 36, range 26-52). Salivary secretions were collected under basal conditions and during masticatory, mechanical, and chemical stimulation before and after four days of cisapride administration (10 or 20 mg four times a day). Cisapride administration resulted in a 45% increase in salivary volume during the basal condition (P < 0.01), a 32% increase during mastication (P < 0.05), a 53% increase during mechanical (P < 0.05), and a 51% increase during chemical (P < 0.01) stimulation. Cisapride administration resulted also in a significant increase in salivary protein output (P < 0.05), salivary bicarbonate (P < 0.05), and nonbicarbonate buffers (P < 0.05), and salivary EGF (P < 0.05). Salivary glycoconjugate significantly increased only during mechanical stimulation with the catheter and at the end of the esophageal perfusion procedure (P < 0.05). Although a similar trend was also recorded during the analysis of salivary PGE2, this difference did not reach statistical significance. Salivary pH and TGF-alpha before and after cisapride administration remained unchanged. The stimulatory impact of cisapride on salivary volume and inorganic (bicarbonate and nonbicarbonate buffers) and organic (protein, glycoconjugate, and EGF) protective components would benefit patients with GERD and would also be potential therapy for xerostomia.
Adult↗
Kainic acid increases the expression of the prohormone convertases furin and PC1 in the mouse hippocampus.
Prohormone convertases (PCs) belong to the mammalian family of subtilisin/kexin-like enzymes which have been implicated in the posttranslational processing of precursor proteins. Several PCs are produced in the central and peripheral nervous system, and only a few specific precursor-substrates have been identified in vivo. In the nervous system, PCs may be involved in intracellular processing of precursors for neuropeptides, hormones and neurotrophic factors, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). To study the interrelationships between the convertases furin, PC1 and PC2, and the neurotrophins NGF, BDNF and NT-3, we compared their mRNA distribution in different tissues. We also examined their expression in the hippocampus of mice undergoing kainic acid-induced seizures. In this experiment, in situ hybridization (ISH) demonstrated that the levels of mRNA for furin, PC1 and BDNF increased maximally at 3 h after kainic acid administration, followed by a decline to normal levels by 96 h. NGF showed small changes, while NT-3 was downregulated with minimal expression levels between 3 to 12 h. Double ISH with radioactively-labeled riboprobes and digoxigenin-labeled riboprobes demonstrated colocalization of furin with NGF and BDNF in the mouse submaxillary gland, and of furin and PC1 with BDNF in the trigeminal ganglion. Based on colocalization studies and evidence of coordinate expression with NGF and BDNF, we suggest the involvement of furin in processing of proNGF, and of both furin and PC1 in processing of proBDNF.
Animals↗
Impact of Helicobacter pylori colonization on immunoreactive epidermal growth factor and transforming growth factor-alpha in gastric juice. Its potential pathogenetic implications.
Epidermal growth factor (EGF), pivotal in mucosal protection, is partly degraded proteolytically at low pH in the gastric milieu; gastric acid secretion, on the other hand, remains influenced by H. pylori colonization. The aim of this study, therefore, was to evaluate the impact of low pH and H. pylori colonization status on immunoreactive EGF and the other member of EGF-family, immunoreactive transforming growth factor-alpha (TGF-alpha). Eighteen patients with nonulcer dyspepsia (NUD) colonized by H. pylori and 55 NUD patients without H. pylori colonization were investigated. Gastric juice samples were aspirated at the beginning of the endoscopy procedure and immediately placed on ice, and their pH was recorded. The measurement of immunoreactive EGF and TGF-alpha was performed using commercially available radioimmunoassays (RIAs) after adjustment of pH to neutral using an assay buffer. Statistical analysis was performed using sigma-Stat for Windows. The concentration of immunoreactive EGF in patients with NUD colonized by H. pylori was 80% lower (P < 0.02) than in those without H. pylori and in both groups immunoreactive EGF was significantly lower when the pH of gastric juice was below 4.0. The concentration of immunoreactive EGF in H. pylori(+) and H. pylori(-) patients was similar when the pH of aspirated gastric juice was above 4.0. However, with gastric juice pH < 4.0, the EGF concentration was 64% lower in H. pylori(+) patients than H. pylori(-) patients (P < 0.05). In general, the concentration of immunoreactive TGF-alpha in gastric juice was unaffected by H. pylori colonization or pH of gastric juice. It is concluded that: (1) significantly lower immunoreactive EGF concentrations in patients with pH below 4.0 indicate that immunoreactive EGF but not immunoreactive TGF-alpha is affected by an acidic gastric milieu; (2) the further reduction of gastric juice immunoreactive EGF at pH below 4.0 in patients colonized by H. pylori suggests that this microorganism may elaborate factors that accelerate its proteolytic degradation or inhibit its rate of synthesis and/or secretion; and (3) this diminished content of immunoreactive EGF at low pH, especially in patients colonized by H. pylori, may facilitate the development and/or progression of mucosal damage.
Adult↗
Enhancement of salivary esophagoprotection: rationale for a physiological approach to gastroesophageal reflux disease.
BACKGROUND & AIMS: Secretion of salivary protective factors in patient s with gastroesophageal reflux disease is impaired. However, the impact of physiological stimulus mastication on salivary protective factors output remains largely unknown. The aim of this study was to measure salivary volume, pH, HCO3-, peptide growth factors, prostaglandin, mucin, protein, and viscosity during mastication. METHODS: In 31 asymptomatic volunteers and 36 patients with endoscopic reflux esophagitis, in basal and parafilm chewing-stimulated saliva, its volume, pH, bicarbonate, epidermal growth factor, transforming growth factor alpha, prostaglandin E2, mucin, protein, and viscosity were investigated. RESULTS: Masticatory stimulation in controls resulted in a significantly increased salivary volume by 205%, pH by 7.6%, bicarbonate by 335%, mucin by 137%, protein by 98%, epidermal growth factor by 123%, and prostaglandin E2 by 132%, accompanied by an increase in transforming growth factor alpha by 80% with 19% decline in viscosity vs. basal values. Mastication in reflux esophagitis significantly increased salivary volume by 215%, pH by 6.8%, bicarbonate by 257%, mucin by 135%, protein by 94%, epidermal growth factor by 207%, and prostaglandin E2 by 240%, whereas transforming growth factor alpha increased by 225% and viscosity by 64% when compared with corresponding basal values. CONCLUSIONS: A profound and significant increase in the secretion rate of inorganic and organic protective components in saliva during masticatory stimulation suggests its potential value as a therapeutic approach to the treatment of patients with gastroesophageal reflux disease.
Adult↗
The isoforms of proprotein convertase PC5 are sorted to different subcellular compartments.
The proprotein convertase PC5 is encoded by multiple mRNAs, two of which give rise to the COOH-terminal variant isoforms PC5-A (915 amino acids [aa]) and PC5-B (1877 aa). To investigate the differences in biosynthesis and sorting between these two proteins, we generated stably transfected AtT-20 cell lines expressing each enzyme individually and examined their respective processing pattern and subcellular localization. Biosynthetic analyses coupled to immunofluorescence studies demonstrated that the shorter and soluble PC5-A is sorted to regulated secretory granules. In contrast, the COOH-terminally extended and membrane-bound PC5-B is located in the Golgi. The presence of a sorting signal in the COOH-terminal 38 amino acids unique to PC5-A was demonstrated by the inefficient entry into the regulated secretory pathway of a mutant lacking this segment. EM of pancreatic cells established the presence of immunoreactive PC5 in glucagon-containing granules, demonstrating the sorting of this protein to dense core secretory granules in endocrine cells. Thus, a single PC5 gene generates COOH-terminally modified isoforms with different sorting signals directing these proteins to distinct subcellular localization, thereby allowing them to process their appropriate substrates.
8-Bromo Cyclic Adenosine Monophosphate↗
Detrimental impact of acid and pepsin on the rate of luminal release of transforming growth factor alpha. Its potential pathogenetic role in the development of reflux esophagitis.
The impact of intraluminal acid and pepsin on the rate of esophageal luminal release of transforming growth factor alpha (TGF alpha), measured by RIA, in 21 asymptomatic volunteers and 26 patients with reflux esophagitis (RE) was investigated. Esophageal secretion was collected, using an esophageal perfusion catheter, during mucosal exposure to NaCl, HCl or HCl/Pepsin and final saline. The basal rate of luminal TGF alpha release in controls was steady throughout the entire four perfusion periods with saline. This rate declined by 71% during mucosal exposure to HCl (p = 0.002) and by 74% during esophageal perfusion with HCl/pepsin (p = 0.011). The basal rate of luminal TGF alpha release in patients with RE was 27% higher than the corresponding value in controls (1.076 +/- 0.140 vs. 0.850 +/- 0.180 ng/min, p = 0.050). Mucosal exposure to acid and acid/pepsin solutions in RE patients also resulted in a significant decline in the luminal release of TGF alpha by 43% (p < 0.001) and by 42% (p < 0.001) respectively. Despite this decline, TGF alpha in patients with RE was significantly higher (p < 0.001) than in controls. The decline in esophageal TGF alpha release during HCl and HCl/pepsin exposure may facilitate the development of mucosal damage. The increase in esophageal TGF alpha release in patients with RE may represent a compensatory mechanism developed by the mucosal inflammatory changes.
Adult↗
Implications of the subtilisin/kexin-like precursor convertases in the development and function of nervous tissues.
Furin, PC1, PC2, and PC5 represent mammalian convertases (PCs) found in endocrine, central and peripheral nervous tissues, which cleave a number of precursors at basic residues normally processed in vivo. Typical bonds cleaved by PCs include the pairs Lys-Arg, Arg-Arg and Arg-X-Lys/Arg-Arg. These cleavage sites have been detected following coexpression of each convertase in cell lines together with different precursors as models, including proopiomelanocortin (POMC), proinsulin and proNGF and proBDNF. The presence of PCs and different precursors was revealed by in situ hybridization or immunocytochemistry in cultured AtT-20 cells, in the developing CNS, pituitary, and pancreatic islets. In an experimental model of epilepsy in which epileptiform activities were provoked by kainic acid administration, we observed a similar transient expression of furin and PC1 as compared to that of NGF and BDNF. In conclusion, it is proposed that under different stimuli various precursors are activated by a unique cocktail of convertases, each of which either alone or in combination with others acts to process inactive precursors, and thereby playing an important role in development and in the plasticity of the neuronal system.
Amino Acid Sequence↗