ARTICLE |
Correspondence to: Richard A. Murphy, Montreal Neurological Institute, 3801 University Ave. Montreal QC H3A 2B4, Canada. Tel.: 514-398-5359; Fax: 514-398-8248.
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Summary |
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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. (J Histochem Cytochem 45:795-804, 1997)
Key Words: prohormone convertase, nerve growth factor, NGF, neurotrophins, PC1, PC2, furin, mouse submandibular glands, immunocytochemistry, in situ hybridization
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Introduction |
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Granular convoluted tubule (GCT) cells within submandibular glands (SGs) of adult mice contain high levels of nerve growth factor (NGF). Production of the protein is controlled by circulating testosterone, and therefore NGF levels elevate at puberty and are higher in SGs of male than of female mice (reviewed by
NGF is a homodimeric polypeptide made up of two 118 amino acid subunits. The monomer arises from the COOH terminus of a 390-amino-acid precursor (pre-pro-NGF) that is cleaved following an Arg-Ser-Lys-Arg sequence immediately preceding the NH2 terminus of the mature protein (
PC1 and PC2 usually cleave precursor molecules at solvent-accessible single or paired basic amino acids. Furin, which cleaves at the consensus site Arg-X-(Lys/Arg)-Arg, processes a number of precursors, including the pro-von Willebrand factor (
Furin has been localized to the constitutive secretory pathway through a membrane-spanning C-terminal domain associated with the trans-Golgi network (
In this study we compared the developmental expression and cellular localization of NGF, PC1, PC2, and furin in mouse SGs. The purpose was to determine whether these convertases are candidates for processing pro-NGF in vivo. The data we obtained are consistent with the idea that furin, but not PC1 or PC2, could generate NGF from its precursor in mouse SGs.
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Materials and Methods |
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Animals
Male and female Balb/c mice obtained from Charles River Breeding Laboratories (St-Constant, Quebec, Canada) were housed in small groups under standard lighting conditions with free access to water and food. Before surgery, animals were anesthetized by IP injections of sodium pentabarbitol (30 mg/kg).
Complementary RNA Probes
Plasmids for the prohormone convertases furin, PC1, and PC2 were prepared and used as described by
Northern Blot Analysis
Total RNA was extracted from mouse SGs using the TRIzol total RNA isolation reagent (Gibco; Grand Island, NY) according to manufacturer's instructions. Samples of RNA were processed as described previously (
In Situ Hybridization
In situ hybridization was performed using the protocol described by
Immunocytochemistry
Rabbit polyclonal antiserum raised against mouse 2.5S NGF (
Detection of Furin by Western Blotting
Golgi fractions of adult male mouse SGs were isolated using a methodology based on the isolation of rat liver Golgi fractions (
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Results |
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Northern Blot Analyses
Northern blot data confirm that SGs of adult male mice contain higher levels of mRNA coding for NGF than glands of 21-day males or adult females (Figure 1A). The differences are due to circulating testosterone which promotes the production of NGF within GCT cells, as reviewed previously (
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The developmental expression of furin and PC2 mRNA differs (Figure 1B). Levels of furin mRNA in SGs of postnatal Day 1 mice are approximately equal in males and females. Levels decrease in females between postnatal Days 1 and 21, and in the adult are lower than in the newborn. In males, furin mRNA levels remain stable throughout postnatal development and are as high in adult glands as in newborns. Levels of PC2 mRNA are comparable in glands from male and female mice on Day 1 but decline with increasing age in both sexes. mRNA coding for PC1 is not detectable in SGs of mice of either sex at any developmental stage (data not shown).
Morphological Studies
The postnatal development of mouse SGs has been well described (
In adult males, GCT cells account for approximately 57% of the SG's volume, acinar cells 23% of volume, and striated and intercalated duct cells together approximately 1% of gland volume. In contrast, GCT cells in glands of adult female mice account for only 19% of SG volume. The remainder of the gland is made up of acinar cells (53% of gland volume) and striated and intercalated duct cells (6% of gland volume) (
In Situ Hybridization
Intense hybridization for PC2 mRNA is evident in small clusters of neurons within parasympathetic ganglia located in intralobular regions of SGs from adult mice (Figure 2) (
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Furin mRNA is widespread. In newborn (Figure 3A) and postnatal Day 10 male mice (Figure 3B), furin mRNA is distributed within the pro-acinar cells and within cells associated with the developing duct system. In 21-day-old and adult animals (Figure 3C and Figure 3D), labeling is evident in duct cells as well as in GCT cells and acinar cells.
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Immunocytochemistry
Figure 4 and Figure 5 compare the developmental appearance of NGF and furin in SGs of male (Figure 4) and female (Figure 5) mice. NGF immunoreactivity is absent from glands of newborn and 10-day mice of both sexes (Figure 4A, Figure 4B, Figure 5A, and 5B) but is evident in GCT cells from glands of 21-day and adult males and females (Figure 4C, Figure 4D, Figure 5C, and Figure 5D). Higher levels of NGF immunoreactivity are present in glands of male mice owing to their content of larger and more numerous GCT cells.
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Unlike NGF, furin immunoreactivity is evident in SGs of both male (Figure 4) and female (Figure 5) mice of all ages tested. In postnatal Day 1 animals (Figure 4E and Figure 5E), immunoreactivity is evident in pro-acinar and duct cells. In SGs from Day 10 animals, furin immunoreactivity remains widespread but is especially concentrated within ductule structures (Figure 4F and Figure 5F). In 21-day and adult animals of both sexes, furin staining is visible within acinar cells (Figure 4G, Figure 4H, Figure 5G, and Figure 5H), but highest levels are evident in duct cells, including GCTs. In some GCT cells, furin immunoreactivity is evident in the apical cell cytoplasm, in a position similar to that of NGF (
Immunoblotting
To confirm that furin protein is present within SGs, we carried out Western blot analysis using the furin-specific monoclonal antibody. Furin immunoreactivity is not detected in crude SG homogenates (Figure 6, Lane 3) but is evident in isolated Golgi fractions, migrating with the expected molecular mass of 100 kD (Figure 6, Lane 4). In these samples, the marker enzyme galactosyl transferase had been concentrated 20-fold compared to the starting homogenate (data not shown). Furin immunoreactivity is not evident in samples blotted with serum not containing the monoclonal antibody (Figure 6, Lanes 1 and 2).
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Discussion |
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The data in this study show that mouse SGs contain PC2 and furin, two members of the convertase family of pro-hormone processing enzymes. In SG from adult mice, furin and NGF co-localize within GCT cells.
mRNA coding for PC2 was detected by Northern blot analyses (Figure 1) in SGs of newborn mice of both sexes; levels were highest in the newborn and declined with increasing age, a result that was explained by in situ hybridization results. PC2 mRNA is present within postmitotic parasympathetic neurons. Therefore, it is not surprising that PC2 mRNA levels in neurons decrease in proportion to total gland mRNA because other cells in the developing SG are dividing, enlarging, and differentiating. Previous studies have localized PC2 in brain neurons (
Because PC1 and PC2 are not present in the same cells as NGF, neither enzyme is a candidate for processing salivary pro-NGF. This result is consistent with in vitro data showing that neither PC1 nor PC2 effectively processes pro-NGF (
Furin is widely distributed within SGs of female and male mice throughout development. In females, furin mRNA levels are highest at birth (Figure 1B), and furin immunoreactivity is evident in pro-acinar and ductule cells (Figure 5). Whether terminal tubules also contain furin is unclear from our studies because these cells are difficult to identify in our sections. As the animals age, levels of furin mRNA decline but furin immunoreactivity remains detectable in acinar cells and ductule cells. In the adult, high levels of furin immunoreactivity were present in GCT cells.
In SGs from male mice, the cellular distribution of furin is similar to that in females but, unlike females, levels of furin mRNA remain high throughout development. These differences probably arise from furin's production in GCT cells, which are larger and more numerous in glands of males. Co-localization of NGF and furin in GCT cells is consistent with experimental evidence showing that furin effectively processes the NGF precursor (
Testosterone does not appear to directly regulate SG production of furin, at least during the early stages of development. Furin mRNA and protein are abundant in SGs of postnatal Day 1 animals, well before pubescent surges in circulating testosterone promote GCT cell development and NGF production. In glands from adult mice, furin mRNA and protein are also detectable in acinar cells and ductule cells, which are not regulated by testosterone. Therefore, furin must carry out additional functions within the SG that are independent of growth factor processing. Testosterone could regulate furin production in GCT cells of pubescent and adult mice through its effects on GCT cells.
Furin has been found in cells that release proteins by constitutive, nonregulated secretory mechanisms. Therefore, it was somewhat surprising to find such high levels of furin mRNA and protein in GCT cells. GCT cells store NGF and other proteins in cellular vesicles that are released by regulated secretory mechanisms (
Furin immunoreactivity was not detected on Western blots of SG homogenates, but was detected in isolated Golgi preparations containing concentrated levels of processed proteins. In contrast, furin immunoreactivity was readily detected by immunocytochemistry. Differences in detection sensitivity may be due to the furin monoclonal antibody reacting more effectively with native furin than with denatured and reduced forms of the protein. Furthermore, we needed to develop tissue sections approximately five times longer to visualize furin by immunocytochemistry than to visualize NGF, which may reflect differences in the affinities of the two antibodies or relative concentrations of furin and NGF within SGs.
Other proteolytic enzymes that can process NGF have been detected within SGs. The first was identified as a component of 7S NGF, a non-covalently linked protein complex in which mature NGF (called ß NGF) is associated with two arginine-specific esterases, the - and
-subunits (
- but not the
-subunit can generate mature NGF from its precursor, but only when used at high stoichiometric levels. For that reason, the physiological relevance of the
-subunit has been questioned (
- and
-subunits have not been detected in other NGF-producing cells and tissues (
Our observation that furin co-localizes with NGF in GCT cells of SGs, taken together with the studies of
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Footnotes |
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* These authors contributed equally to this work.
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Acknowledgments |
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Supported by Medical Research Council of Canada Program Grant MRC-PG11473 and by the Canadian Network of Centers of Excellence (NCE) on Neural Regeneration and Functional Recovery (to RAM). SP was supported by a NCE fellowship and RD is a fellow of the Fonds de la Recherche en Santé du Québec.
We thank Jacynthe Laliberté, Xue-Wen Yuan, and Ali Fazel for excellent technical assistance. We also thank Dr W.J. van de Ven for providing us with the furin antibody and Dr Gary Thomas for the vector coding for the furin fragment.
Received for publication August 1, 1996; accepted January 6, 1997.
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