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Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate.
ABSTRACT: Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.
Project description:AIM:Periodontitis induced by oral pathogens leads to severe periodontal tissue damage and osteoclast-mediated bone resorption caused by inflammation. On the basis of the importance of Ac45 in osteoclast formation and function, we performed this study to evaluate the therapeutic potential of periodontitis by local adeno-associated virus (AAV)-mediated Ac45 gene knockdown. MATERIAL AND METHODS:We used AAV-mediated short hairpin RNAi knockdown of Ac45 gene expression (AAV-sh-Ac45) to inhibit bone erosion and gingival inflammation simultaneously in a well-established periodontitis mouse model induced by Porphyromonas gingivalis W50. Histological studies were performed to evaluate the bone protection of AAV-sh-Ac45. Immunochemistry, ELISA and qRT-PCR were performed to reveal the role of Ac45 knockdown on inflammation, immune response and expression of cytokine. RESULTS:We found that Ac45 knockdown impaired osteoclast-mediated extracellular acidification and bone resorption in vitro and in vivo. Furthermore, local administration of AAV-sh-Ac45 protected mice from bone erosion by >85% and attenuated inflammation and decreased infiltration of T cells, dendritic cells and macrophages in the periodontal lesion. Notably, the expression of pro-inflammatory cytokines was also reduced. CONCLUSIONS:Local AAV-sh-Ac45 gene therapy efficiently protects against periodontal tissue damage and bone erosion through both inhibition of osteoclast function and attenuating inflammation, and may represent a powerful new treatment strategy for periodontitis.
Project description:Vacuolar-type H(+)-ATPases (V-ATPases) are macromolecular proton pumps that acidify intracellular cargos and deliver protons across the plasma membrane of a variety of specialized cells, including bone-resorbing osteoclasts. Extracellular acidification is crucial for osteoclastic bone resorption, a process that initiates the dissolution of mineralized bone matrix. While the importance of V-ATPases in osteoclastic resorptive function is well-defined, whether V-ATPases facilitate additional aspects of osteoclast function and/or formation remains largely obscure. Here we report that the V-ATPase accessory subunit Ac45 participates in both osteoclast formation and function. Using a siRNA-based approach, we show that targeted suppression of Ac45 impairs intracellular acidification and endocytosis, both are prerequisite for osteoclastic bone resorptive function in vitro. Interestingly, we find that knockdown of Ac45 also attenuates osteoclastogenesis owing to a reduced fusion capacity of osteoclastic precursor cells. Finally, in an effort to gain more detailed insights into the functional role of Ac45 in osteoclasts, we attempted to generate osteoclast-specific Ac45 conditional knockout mice using a Cathepsin K-Cre-LoxP system. Surprisingly, however, insertion of the neomycin cassette in the Ac45-Flox(Neo) mice resulted in marked disturbances in CNS development and ensuing embryonic lethality thus precluding functional assessment of Ac45 in osteoclasts and peripheral bone tissues. Based on these unexpected findings we propose that, in addition to its canonical function in V-ATPase-mediated acidification, Ac45 plays versatile roles during osteoclast formation and function.
Project description:PC2 is a neuroendocrine endoprotease involved in the processing of prohormones and proneuropeptides. PC2 is synthesized as a proenzyme which undergoes proteolytic maturation within the cellular secretory apparatus. Cleavage occurs at specific sites to remove the N-terminal propeptide. The aim of the present study was to investigate structural requirements for the transfer of proPC2 through the secretory pathway. A series of mutant proPC2 constructs were transfected into COS-7 cells and the fate of the expressed proteins followed by pulse-chase analysis and immunocytochemistry. Human PC2 was secreted relatively slowly, and appeared in the medium primarily as proPC2 (75 kDa), together with much lower amounts of a processed intermediate (71 kDa) and mature PC2 (68 kDa). Mutations within the primary processing site or the catalytic triad caused the protein to accumulate intracellularly, whereas deletion of part of the propeptide, the P-domain or the C-terminal regions also prevented secretion. Immunocytochemistry showed that wild-type hPC2 was localized mainly in the Golgi, whereas two representative mutants showed a distribution typical of proteins resident in the endoplasmic reticulum. The results suggest that proenzyme processing is not essential for secretion of PC2, but peptides containing mutations that affect the ability of the propeptide (and cleavage sites) to fold within the catalytic pocket are not transferred beyond the early stages of the secretory pathway. C-terminal sequences may be involved in stabilizing such conformations.
Project description:Lysosomal trafficking and protease exocytosis in osteoclasts are essential for ruffled border formation and bone resorption. Yet the mechanism underlying lysosomal trafficking and the related process of exocytosis remains largely unknown. We found ATP6ap1 (Ac45), an accessory subunit of vacuolar-type H(+)-ATPases (V-ATPases), to be highly induced by receptor activator for nuclear factor kappa B ligand (RANKL) in osteoclast differentiation. Ac45 knockdown osteoclasts formed normal actin rings, but had severely impaired extracellular acidification and bone resorption. Ac45 knockdown significantly reduced osteoclast formation. The decrease in the number of osteoclasts does not result from abnormal apoptosis; rather, it results from decreased osteoclast precursor cell proliferation and fusion, which may be partially due to the downregulation of extracellular signal-regulated kinase (ERK) phosphorylation and FBJ osteosarcoma oncogene (c-fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), and "transmembrane 7 superfamily member 4" (Tm7sf4) expression. Notably, Ac45 knockdown osteoclasts exhibited impaired lysosomal trafficking and exocytosis, as indicated by the absence of lysosomal trafficking to the ruffled border and a lack of cathepsin K exocytosis into the resorption lacuna. Our data revealed that the impaired exocytosis is specifically due to Ac45 deficiency, and not the general consequence of a defective V-ATPase. Together, our results demonstrate the essential role of Ac45 in osteoclast-mediated extracellular acidification and protease exocytosis, as well as the ability of Ac45 to guide lysosomal intracellular trafficking to the ruffled border, potentially through its interaction with the small guanosine-5'-triphosphatase (GTPase) Rab7. Our work indicates that Ac45 may be a novel therapeutic target for osteolytic disease.
Project description:To observe the change of the neuropeptide pro-protein processing system in the ischemic retina ganglion cell-5 (RGC-5) cells, pro-protein convertase-2 (PC2), carboxypeptidase-E (CPE) and preproneuropeptide Y (preproNPY) protein levels in the ischemic RGC-5 cells and conditioned medium were analyzed.The RGC-5 cell was differentiated in 0.1 mumol/L staurosporine for 24 h and then stressed by different doses of oxygen and glucose deprivation (OGD). The acute or chronic OGD-induced cell death rates were obtained by using PI or TUNEL staining. The protein expression levels were determined by using the Western blot method and PC2 activity analysis.The ischemia caused substantial cell death in an OGD dose-dependent manner. In the cells, proPC2 and preproNPY protein levels gradually increased whereas proCPE gradually decreased. After OGD, PC2 activity was decreased. In the conditioned medium, proPC2 and PC2 proteins gradually decreased whereas proCPE, CPE, and preproNPY proteins gradually increased.These results demonstrated that OGD inhibited the neuropeptide pro-protein processing system by reducing PC2 activity and the maturation of proPC2. The aggregation of the pro-proteins and the increase of the active CPE excision adversely exacerbated the cell injury. The pro-protein processing system might play a critical role in the ischemic stress of RGC-5 cells.
Project description:Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c'', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c'', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.
Project description:The role of PC2 in prosomatostatin (PSS) processing was investigated in GH3/GH4C1 pituitary cells. These cells are sparsely granulated, express different amounts of PC2 and no PC1. We described heterologous processing of rat PSS (rPSS) co-expressed with PC2 in stably transfected cells, correlate PC2 protein levels under different conditions of transfection with efficiency of PSS processing to somatostatin-14 (SS-14), determine the effect of modulating cell granularity on enzyme expression and PSS processing, and compare the relative potency of PC2 with that of PC1, PSS and cleavage products were monitored by HPLC and radioimmunoassay of SS-like immunoreactivity (SSLI). Radioimmunoassay analysis of N-terminal PC2-like immunoreactivity (PC2 LI) in GH4C1:rPSS, GH4C1:rPSS + PC2 and GH3:rPSS transfectants showed a gradient of PC2 protein of 1:2.6:3.4 in cell extracts and 1:4.7:9 in secretion media from these cells respectively. The concentration of PC2 protein correlated with SS-14 conversion efficiency was 36 +/- 3% in GH4C1:rPSS cells, 56 +/- 7% in GH4C1:rPSS-PC2 cells and 100% in GH3:rPSS cells. Treatment of GH4C1:rPSS + PC2 cells with epidermal growth factor, insulin, and beta-estradiol to induce granules, significantly increased basal and forskolin-stimulated co-release of SS LI and PC2 LI, but had no influence on SS-14 processing efficiency. Hormone treatment led to a small increase in the ratio of mature PC2 (68 kDa) to proPC2 (75 kDa) forms. PC1 stably transfected in GH4C1 cells produced significantly greater SS-14 conversion (62% in cells, 66% in media) compared with PC2 transfectants (53% in cells, 47% in media) These results provide the first proof that PC2 can effect dibasic processing of mammalian PSS, and, along with PC1, qualifies as an authentic SS-14 convertase. The activity of PC2 requires the milieu of the secretory cell but not the secretory granule.
Project description:Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggregation and membrane association during its transit through the regulated secretory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate this further, we determined the binding properties of a peptide containing amino acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enriched membranes from the AtT20 cell line. Removal of peripheral membrane proteins or hydrolysis of integral membrane proteins did not affect the binding properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca(2+)-dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we used chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depleting detergent, failed to extract PC2 from the membranes, whereas chromogranin A (CgA) was removed. Treatment of the membranes with Triton X-100 yielded a low-density detergent-insoluble fraction enriched in PC2, but not CgA. The detergent-insoluble fraction also contained glycoprotein III, known to be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid rafts in the membrane and its sorting into the regulated secretory pathway.
Project description:The vacuolar (H(+))-ATPase (V-ATPase) is the main regulator of intraorganellar pH and in neuroendocrine cells is controlled by its accessory subunit, Ac45. Here, we report the discovery of the first isoform of a V-ATPase accessory subunit, namely an Ac45-like protein, denoted Ac45LP. Phylogenetic analysis revealed a lineage-dependent evolutionary history: Ac45 is absent in birds, and Ac45LP is absent in placental mammals, whereas all other tetrapod species contain both genes. In contrast to Ac45, Ac45LP is not proteolytically cleaved, a prerequisite for proper Ac45 routing. Intriguingly, Xenopus Ac45LP mRNA was expressed in developing neural tissue and in neural crest cells. In adult Xenopus, Ac45 mRNA is widely expressed mostly in neuroendocrine tissues, while Ac45LP mRNA expression was found to be restricted to the kidney and the lung. This novel Ac45LP may provide additional possibilities for V-ATPase regulation during neurodevelopment as well as in kidney and lung cells.
Project description:Insulin secretion from pancreatic beta cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated beta cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of beta cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3(-/-) mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3(-/-) beta cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3(-/-) mice, while in Clcn3(+/+) cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic beta cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.