Furin mRNA Research Articles

BetaAPP and furin mRNA concentrates in immature senile plaques in the brain of Alzheimer patients.

This study examined the possibility that in Alzheimer disease (AD) beta-amyloid precursor protein (betaAPP) mRNA is delivered to senile plaques (SPs) via dendritic processes. BetaAPP mRNA was detected in SPs by in situ hybridization, using a 1.4-kb cRNA in which both [35S]-UTP and [35S]-CTP were incorporated together. The betaAPP mRNA was compared with that of furin, a proteolytic enzyme putatively involved in betaAPP processing, and its orthologue proprotein convertase PCI served as a control. Human presenile AD cases with mostly immature SPs and AD cases generally with mature SPs were analyzed. To decrypt SPs after hybridization, brain sections were stained with thioflavin S. To establish relationships between the density of dystrophic fibers, the degree of plaque maturation, and the concentration of mRNA in SPs, the plaque maturity markers Abeta(1-42) and Abeta(1-40) peptides were co-localized with neurofilament protein 200 and compared with microtubule-associated protein 2 (MAP 2). The results suggest that immature, Abeta(1-42)- and dystrophic dendrite-containing SPs (but not mature SPs containing Abeta(1-40) and missing dystrophic dendrites) are capable of concentrating specific mRNAs. Dystrophic dendrites may thus serve as a route for the transport of specific mRNAs from the cell bodies to SPs.

The proprotein convertase PACE4 is upregulated by PDGF-BB in megakaryocytes: gene expression of PACE4 and furin is regulated differently in Dami cells.

The differentiation of megakaryocytes into platelets is highly regulated by many cytokines and growth factors. PACE4 and furin are Ca(2+)-dependent serine endoproteases belonging to the subtilisin-like proprotein convertase (SPC) family. These enzymes are involved in the proteolytic activation of proteins that play essential roles in cell growth and differentiation. In this study, we examined the expression of PACE4 and furin during the differentiation of megakaryoblastic cell lines, Dami and HEL cells, induced by phorbol 12-myristate 13-acetate (PMA). PMA stimulates not only the expression of platelet-derived growth factor-B (PDGF-B) mRNA, but also PACE4 mRNA in these cell lines. The expression of PACE4 transcripts (both the PACE4A and PACE4C/CS isoforms) was upregulated more than 4-fold by PMA. Moreover, direct treatment with PDGF-BB also resulted in an increase in the level of PACE4 mRNA. Further, the effect of PDGF-BB on PACE4 expression was confirmed by promoter assay of the PACE4 gene. Although the furin mRNA level was increased by TGF-beta1 in Dami cells, it was not affected by PDGF-BB. These results indicate for the first time that PACE4 expression is specifically upregulated by PDGF-BB in differentiated megakaryoblasts, suggesting a unique role for PACE4 in platelet production.

Prohormone convertase furin has a role in gastric cancer cell proliferation with parathyroid hormone-related peptide in a reciprocal manner.

We investigated the expression of parathyroid hormone-related peptide (PTHrP) and the relationship between PTHrP and its endoprotease furin in gastric cancer. PTHrP was colocalized with furin in 75% of gastric cancer tissues (six of eight) from patients with high serum PTHrP levels. PTHrP mRNA expression was confirmed in 67% of gastric cancer cell lines (four of six), whereas furin mRNA was detected in all six gastric cancer cell lines. In a cultured gastric cancer cell line, MKN28, mature PTHrP protein expression was markedly increased by transfection of furin cDNA. Furin cDNA-transfected MKN28 cells grew faster than did the mock controls. Moreover, furin mRNA expression in cultured gastric cancer cells was enhanced when PTHrP was added to the culture medium. These results suggest a link between PTHrP and furin in the regulation of gastric cancer cell growth. Furin might be involved not only in the production of the mature form of PTHrP, but also in promoting growth in gastric cancer cells.

Elevated furin expression in aggressive human head and neck tumors and tumor cell lines.

Pro-protein convertases (PCs) are proteases that recognize and cleave precursor proteins. Furin, a well-studied PC, is ubiquitously expressed, and it has been implicated in many physiological and pathological processes. Some substrates for furin, such as membrane type 1 (MT1) matrix metalloproteinase (MMP), an MMP that activates gelatinase, a collagen-degrading enzyme, are associated with the advanced malignant phenotype. This report examines the expression of furin in carcinoma cell lines of different invasive ability. The levels of furin mRNA and protein correlated with the aggressiveness of tumor cell lines derived from head and neck and lung cancers. Furin expression also was investigated in primary head and neck squamous cell carcinomas (HNSCCs). Furin mRNA was not detected in nonmetastasizing carcinomas. In contrast, furin mRNA was expressed in metastasizing HNSCCs. Immunohistochemistry and Western blot analysis confirmed these results at the protein level. Furin activity was investigated indirectly by evaluating the expression of the pro-form and the processed form of MT1-MMP. Metastasizing HNSCCs showed increased expression of MT1-MMP. Furthermore, pro-MT1-MMP expression was noted in most of the nonmetastasizing HNSCCs analyzed by Western blot, and it was absent in the metastasizing HNSCCs. This finding suggests a lower level of furin-mediated MT1-MMP activation in the less aggressive cancers. These observations indicate that furin plays a role in tumor progression. Its overexpression in more aggressive or metastasizing cancers resulted in increased MMP processing.

Furin and membrane type-1 metalloproteinase mRNA levels and activation of metalloproteinase-2 are associated with arterial remodeling

Matrix metalloproteinase (MMP) activation is an essential feature of pathological and physiological arterial enlargement or shrinkage. Recently, furin-activated membrane type-1 MMP (MT1-MMP) was identified as the in vivo activator of MMP2 in mice. Although arterial enlargement and shrinkage are important in several pathological processes, this proprotein convertase–MT1-MMP axis has not been described during arterial remodeling. In rabbit femoral and carotid arteries, we report an increase in furin and MT1-MMP mRNA levels before and at the onset of arterial remodeling followed by an increase in activated MMP2. This reveals the presence of the proprotein convertase–MT1-MMP axis in flow-induced arterial remodeling and identifies furin as a possible target for local intervention in pathological arterial remodeling.

Processing of immunosuppressive pro-TGF-beta 1,2 by human glioblastoma cells involves cytoplasmic and secreted furin-like proteases.

TGF-beta is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-beta processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-beta or neutralizing TGF-beta Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-beta1 and TGF-beta2 precursor molecules and, consequently, the release of mature bioactive TGF-beta molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-beta processing, and TGF-beta release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-beta bioactivity.

Upregulatory Expression of Furin and Transforming Growth Factor-β by Fluid Shear Stress in Vascular Endothelial Cells

Furin, a yeast Kex2-family endoprotease, converts many vasoregulatory propeptides, including pro-transforming growth factor (TGF)-beta to their mature forms. We examined whether furin expression is regulated by shear stress in vivo and in vitro. When an arteriovenous shunt was placed between the carotid artery and external jugular vein in rabbits, furin and TGF-beta were highly expressed in shear stress-loaded endothelial cells. Exposure of bovine aortic endothelial cells in culture to shear stress induced furin and TGF-beta expression in a similar manner. Molecular analysis of furin expression in bovine aortic endothelial cells revealed that shear stress increases the furin gene expression at transcriptional levels. Furthermore, TGF-beta itself increased the furin mRNA levels. Shear-mediated furin expression was partly mediated by TGF-beta because shear-induced furin mRNA levels were considerably decreased by overexpression of the truncated form of the TGF-beta type II receptor. Likewise, blockade of furin activity by a furin inhibitor significantly decreased the endothelial production of mature TGF-beta. Taken together, the results indicate that furin expression is induced and maintained by a coordination of shear stress and TGF-beta. Increased furin expression may facilitate the formation of mature TGF-beta, resulting in the enhanced effects of TGF-beta on endothelial cells and vascular smooth muscle cells in the vasculature.

Furin-independent Pathway of Membrane Type 1-Matrix Metalloproteinase Activation in Rabbit Dermal Fibroblasts

We investigated the gene expression and intracellular activity of processing protease furin and its involvement in the process of membrane type 1-matrix metalloproteinase (MT1-MMP) activation in rabbit dermal fibroblasts. When the rabbit fibroblasts were treated with concanavalin A (ConA), pro-MMP-2 was converted to an active 62-kDa MMP-2 through the appearance of a 64-kDa intermediate MMP-2. The ConA-induced pro-MMP-2 activation resulted from increasing the gene expression and production of MT1-MMP in the rabbit fibroblasts. Reverse transcriptase-polymerase chain reaction demonstrated that in rabbit dermal fibroblasts furin mRNA was detected and, unlike MT1-MMP, was not increased by ConA. These findings are further supported by the fact that the intracellular furin activity also was constitutively detected and was unchanged by the ConA treatment. Very similar phenomena were also observed in human uterine cervical fibroblasts, which are known to produce MT1-MMP by ConA stimulation. These results suggest that the expression of the furin gene and the intracellular activity are not regulated by ConA. On the other hand, neither a synthetic furin inhibitor, decanoyl-RVKR-CH(2)Cl (25-100 microM) nor a furin antisense oligonucleotide (40 microM) inhibited the MT1-MMP-mediated pro-MMP-2 activation in ConA-treated rabbit dermal fibroblasts, whereas these compounds interfered with pro-MMP-2 activation in ConA-treated human uterine cervical fibroblasts. Nonetheless, the furin antisense oligonucleotide completely suppressed furin gene expression in both rabbit and human fibroblasts. These results suggest that furin does not participate in the process of MT1-MMP activation induced by ConA in rabbit dermal fibroblasts.

Differentiation-Induced Insulin Secretion from Nonendocrine Cells with Engineered Human Proinsulin cDNA

To investigate the effects of differentiation on insulin secretion from engineered nonendocrine cells, modified human proinsulin cDNA (INS/fur) was transfected to THP-1 monocyte and C2C12 myoblast cell lines. When THP-1 was differentiated into macrophages with phorbol ester, the insulin secretion rate was increased by 3.1-fold. This increase in insulin secretion is accompanied by a 17.6-fold increase in the processing efficiency of the modified human proinsulin and by a 3.5-fold increase in the abundance of furin mRNA. In addition, differentiation of C2C12 into myotubes, which can be induced by changing the serum, showed a 9.9-fold increase in insulin secretion and was accompanied by a 1.6-fold increase in the abundance of furin mRNA. The involvement of posttranslational processing and the exocytotic process in differentiation-induced insulin secretion could lead to the possibility of regulation of insulin secretion from genetically engineered cells.

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells.

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC 7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF.

Co-expression of the proprotein-processing endoprotease furin and its substrate transforming growth factor β1 and the differentiation of rat hepatocytes

Furin, a member of the yeast Kex2 endoprotease family, converts a number of proproteins to their active forms. The liver produces a number of proproteins having a furin-cleavable site; thus, furin may be involved in growth and differentiation both in the partially hepatectomized liver and in primary cultured hepatocytes. Furin mRNA levels are elevated in tissues regenerated from partially hepatectomized rat liver. In primary culture of rat hepatocytes, furin expression increases gradually with time, and its expression is greatly enhanced by transforming growth factor β1, whose processing from the precursor requires cleavage by furin. Thus, we suggest that the regeneration and differentiation of hepatocytes is dependent upon the co-elevation of furin and transforming growth factor β1 mRNAs.

Pro-protein convertase gene expression in human breast cancer.

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PCI and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, proprotein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis.

Pro‐protein convertase gene expression in human breast cancer

As a first step towards elucidating the role that pro-protein convertases play in the growth regulation of breast cancer, we studied the gene expression of 6 known human convertase members (PC1/PC3, PC2, furin/PACE, PACE4, PC5/PC6 and PC7/LPC) in human breast cancer tumors and cell lines. PC1, furin, PACE4 and PC7 mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification in all 7 human breast cancer cell lines and 30 breast tumor tissues tested. PC5 expression was detected in 2/30 tumor tissues. PC2 mRNA, however, was not detected. In situ hybridization localized furin mRNA to the tumor cells; adjacent fibrous stroma and blood vessel elements were negative for furin gene expression. Thirty breast tumors with varying quantities of estrogen and progesterone receptors were assayed for furin, PACE4 and PC1 mRNAs by quantitative RT-PCR, and 22 tumors were assayed for PC7 mRNA. An apparent association was observed only between PACE4 and estrogen receptors. No statistically significant correlation was found between the levels of steroid receptors and the expression of human furin, PC1 and PC7 genes. Convertase mRNA levels appeared similar in both the estrogen-responsive and -unresponsive breast cancer cell lines. Also, pro-protein convertase mRNAs were not detected in 9 histologically normal human breast tissues. These results suggest that elevated expression of some members of the pro-protein convertase gene family is a characteristic of human breast cancer, an event which may be important for human breast tumorigenesis.Int. J. Cancer 71: 966-971, 1997. © 1997 Wiley-Liss Inc.

Prohormone convertases in mouse submandibular gland: co-localization of furin and nerve growth factor.

Nerve growth factor (NGF) in mouse submandibular glands (SGs) is generated from a 35-kD precursor by proteolytic enzymes that have yet to be identified. Prohormone convertases (PCs) cleave the NGF precursor in vitro, and in this study we questioned whether PCs could process salivary NGF in vivo. mRNA coding for PC2 (but not PC1) was detected on Northern blots of SG mRNA and also by in situ hybridization within parasympathetic neurons of intralobular ganglia. Northern blot and in situ hybridization analyses also detect mRNA coding for furin. In SGs of male mice, furin mRNA levels are high at birth and remain high throughout development. In glands from female mice, levels decline during postnatal development and are lower in adults than in newborns. Immunocytochemistry detects furin immunoreactivity in pro-acinar and ductal cells of glands from newborn and pubescent mice. In glands of adults, furin immunoreactivity is detectable in acinar cells but highest levels are present in NGF-containing granular convoluted tubule cells. These data, taken together with those from previous studies, suggest that furin is a candidate processing enzyme for NGF in mouse submandibular glands.

TGFbeta1 regulates gene expression of its own converting enzyme furin.

TGFbeta1 is known for its potent and diverse biological effects, including immune regulation, and cell growth and differentiation. We have recently shown that TGFbeta1 precursor is processed by human furin COOH-terminal to the R-H-R-R278 cleavage site to generate authentic mature TGFbeta1. In the present study, we demonstrate that steady-state furin mRNA levels are increased in rat synovial cells by 2 and 20 ng/ml TGFbeta1. Stimulation with TGFbeta1 results in a significant increase in furin mRNA levels, starting at 3 h with the peak effect observed at 12 h (2.5-fold increase +/-0.4). TGFbeta1 did not increase furin mRNA stability, and treatment of synovial cells with actinomycin D, before TGFbeta1 addition prevented the increase in fur gene expression, suggesting that the observed regulation occurs at the level of gene transcription. Treatment of synovial and NRK-49F fibroblastic cells with exogenous TGFbeta1 (5 ng/ml) or TGFbeta2 (10 ng/ml) translates into an increase in pro-TGFbeta1 processing as evidenced by the appearance of a 40-kD immunoreactive band corresponding to the TGFbeta1 NH2-terminal pro-region. Furin processing activity stimulated by TGFbeta2 correlates with significant increase in extracellular mature and heat-activable TGFbeta1 as determined by an isoform-specific ELISA assay. Taken together, these results demonstrate for the first time that TGFbeta1 upregulates gene expression of its own converting enzyme, and that this expression is translated into augmented processing of the TGFbeta1 precursor form. Such adaptive responsiveness of the TGFbeta1 convertase may represent an important aspect of TGFbeta1 bioavailibility in TGFbeta1-related processes and pathological conditions.

Comparative functional role of PC7 and furin in the processing of the HIV envelope glycoprotein gp160

The intracellular proteolytic processing of HIV envelope glycoprotein gp160 into gp120/gp41 is an essential step for virus infectivity. Several convertases, belonging to the pro-protein convertase family, have been proposed as candidate gp160 processing enzymes. Here we demonstrate using RT-PCR that resting human T4 lymphocytes weakly express PC7, furin, and PC5 mRNA whereas lymphocytes activated under conditions favoring HIV replication express 5–10-fold higher levels of furin and PC7. In this report, we examined the capability of the newly cloned convertase PC7 to cleave gp160 into gp120/gp41 and compared it to furin. This was carried out in a cell-based assay whereby both gp160 and the cognate convertase were co-expressed in the constitutively secreting BSC40 cells and in the regulated AtT20 cells, as well as using two in vitro assays which examined the cleavage of gp160 or of a synthetic peptide spanning the cleavage site. The data demonstrate that PC7 can cleave specifically and in a cell-type specific manner gp160 into gp120/gp41, suggesting that both furin and PC7 are so far the major PC-like candidate gp160 convertase in T4 lymphocytes. © 1997 Federation of European Biochemical Societies.

Co-elevation of brain natriuretic peptide and proprotein-processing endoprotease furin after myocardial infarction in rats

We investigated the expression of the yeast Kex2 family endoproteases furin and PACE4, and brain natriuretic peptide (BNP) in the atrium and ventricle after infarction as well as the conversion of the BNP precursor γBNP to BNP-45. In a rat heart failure model, plasma BNP rose in two phases – first at day 3, and again at day 14. BNP mRNA, as measured by Northern blot analysis, increased strongly at day 3, then at days 14 and 28 less strongly in the atrium, and in the ventricle it increased weakly at day 3, then strongly at days 14 and 28. Furin mRNA showed the same pattern of expression as that of BNP message, whereas PACE4 message stayed unchanged after the infarction. Both furin and BNP were immunostained in the myocardium adjacent to the infarcted tissue. We suggest that after myocardial infarction, furin is co-expressed with BNP in both the atrium and ventricle, and that furin may be responsible for the conversion of γBNP to BNP-45.

Differential modulation of prohormone convertase mRNA by second messenger activators in two cholecystokinin-producing cell lines.

Regulation of prohormone convertase expression was studied in two neuropeptide-producing cell lines, the human neuroepithelioma cell line SK-N-MCIXC and the rat medullary thyroid carcinoma cell line WE 4/2. The cells were treated with the phosphodiesterase inhibitor isobutyl-methylxanthine and the tumor-promoting phorbol ester, phorbol-12-myristate-13 acetate, activators of the cyclic AMP (cAMP) and protein kinase C (PKC) second messenger pathways, respectively. mRNA levels of prohormone convertase 1 (PC1), prohormone convertase 2 (PC2), and furin were determined after 3- and 6-h treatments, using Northern analysis. Activation of both cAMP and PKC pathways increased PC1, but not PC2 or furin mRNA levels in SK-N-MCIXC cells. Activation of the cAMP pathway increased PC1, PC2, and furin mRNA levels in WE 4/2 cells, whereas activation of the PKC pathway did not change prohormone convertase mRNA levels in this cell line. These results indicate that prohormone convertases may be differentially regulated by cAMP and PKC mechanisms and regulation may be tissue specific.

Inhibition of processing of parathyroid hormone-related peptide by anti-sense furin: effect in vitro and in vivo on rat Leydig (H-500) tumor cells.

To attain full biological activity, the precursor pro-parathyroid hormone-related peptide (ProPTHRP) must be converted to the mature peptide PTHRP. We have examined the effect of inhibiting expression of the pro-hormone convertase furin in H-500 rat Leydig tumor cells on PTHRP production and action in vitro and in vivo. H-500 Leydig tumor cells were stably transfected with a mammalian expression plasmid containing furin cDNA in an anti-sense orientation. This resulted in inhibition of endogenous furin mRNA expression and of protein production as assessed by immunocytochemistry. These experimental cells secreted extended NH2-terminal PTHRP forms with reduced adenylate cyclase-stimulating activity. This was associated with a marked decrease in the proliferation of these tumor cells in vitro. Transfected and control cells were then implanted into male Fischer rats. Animals implanted with control cells became hypercalcemic. In contrast, animals implanted with experimental cells maintained near normal levels of plasma calcium. Experimental cells inoculated in vivo developed into tumors of significantly decreased volume compared to control cells and animal survival time was prolonged. Our results indicate that alteration of the processing of PTHRP can diminish the hypercalcemic endocrine actions of PTHRP and can reduce autocrine/paracrine effects of PTHRP on tumor cell growth both in vitro and in vivo. Furin may also exert a broader role in processing other factors required for tumor proliferation. Consequently, anti-sense modulation of furin activity may be a potential modality for understanding the mechanism of neoplastic growth and progression.

Age-related alterations in the expression of prohormone convertase messenger ribonucleic acid (mRNA) levels in hypothalamic proopiomelanocortin mRNA neurons in the female C57BL/6J mouse.

POMC processing is mediated by the prohormone convertases (PC1 and PC2). The cleavage of beta-endorphin-(1-31) is mediated by PC2. PC2 can also further cleave beta-endorphin-(1-31) to beta-endorphin-(1-27). We previously reported a significant increase in the proportion of beta-endorphin-(1-27) and -(1-27) forms in the arcuate nucleus (ARC) of the hypothalamus in middle-aged females with irregular estrous cycles (5-7 days) compared to young female C57BL/6J mice with regular cycles (4-5 days). Changes in processing enzymes may be a mechanism underlying this change. We compared ARC messenger RNA (mRNA) levels of PC1, PC2, and furin by Northern blot and in situ hybridization analyses in young, middle-aged, and old mice. Antisense complementary RNA probes to mouse PC1, PC2, and furin were radiolabeled and used in single label studies, alone or in combination with a mouse POMC digoxigenin-labeled complementary RNA probe for double label studies. For Northern blot analysis, young (4- to 5-month-old) normally cycling (4-5 days) mice at diestrus were compared to middle-aged (12- to 13-month-old) irregularly cycling (5-7 days) mice at diestrus. By Northern blot analysis, a significant increase (P < 0.05) in ARC PC2 mRNA levels was detected in middle-aged compared to young mice, but ARC PC1 and furin mRNA levels were unaltered. Single label in situ hybridization analysis confirmed these findings in the general neuron population. We also observed a significant reduction in ARC furin mRNA levels in old mice compared to either young or middle-aged mice. Double labeling in situ hybridization histochemistry demonstrated that PC2 mRNA levels were significantly increased (at least 2-fold) in POMC mRNA-containing neurons of middle-aged compared to young mice. Selective changes in PC2 mRNA levels in ARC POMC neurons are correlated with changes in beta-endorphin-(1-31) processing to beta-endorphin-(1-27)/(1-26) in middle-aged animals. Our data suggest that the natural age-related shift in beta-endorphin peptide processing is mediated by PC2.