ARTICLE |
Correspondence to: Andrew Bateman, Room L.2.05, Endocrine Laboratory, Royal Victoria Hospital, 687 Pine Ave West, Montréal, Québec H3A 1A1, Canada. E-mail: bateman@rvhmed.lan.mcgill.ca
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Summary |
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Granulins, also called epithelins, are 6-kD peptides with growth modulatory effects on a variety of cells. The granulin/epithelin precursor supports tumorigenesis in appropriate cell models and is the only growth factor able to overcome the cell cycle block that occurs in murine fibroblasts after deletion of a functional IGF-1 receptor. However, little is known of the role of granulin/epithelin gene products in vivo. To understand the physiological role of granulins it is essential to know the cell types and conditions in which it is expressed. We examined granulin/epithelin gene expression in adult rodents by in situ hybridization. The granulin/epithelin precursor is constitutively expressed in a number of epithelia, particularly in the skin, GI tract, and reproductive system. Other epithelia express the gene less strongly. Progranulin is expressed in immune cells in vivo and in specific neurons in the brain, including Purkinje cells, pyramidal cells of the hippocampus, and some cerebral cortical neurons. Little expression was detected in muscle cell, connective tissue, or endothelium. Cumulatively, these results define the basal gene expression of a new growth factor system and suggest that the progranulin/epithelin gene is multifunctional, with important constitutive roles in epithelial homeostasis, reproductive, immunological, and neuronal function. (J Histochem Cytochem 48:9991009, 2000)
Key Words: progranulin, epithelin, acrogranin, PCDGF, TGFe, growth factor
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Introduction |
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Granulins (grns) (
Although progranulin, progranulin-derived peptides, and related proteins such as TGFe are clearly capable of regulating cell growth, less is known of their roles in vivo. Most established cell lines of epithelial origin express the progranulin gene strongly (
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Materials and Methods |
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Isolation of RNA and Northern Blotting Analysis
Around 2 x 106 cells were harvested and washed with ice-cold PBS, pH 7.4, Total cellular RNA was isolated by the guanidium thiocyanate method (
Generation of Digoxigenin-UTP-labeled Progranulin RNA Probe
A 238-bp fragment corresponding to nucleotides 7481002 of full-length rat progranulin cDNA was blunt end-ligated to an expression vector BlueScript KII (Invitrogen, Carlsbad, CA). The 5' end of the fragment is adjacent to an Xba restriction site with an upstream T7 promoter; a Xho restriction site is located downstream of the 3' end of the fragment with a T3 promoter further down on the line. Sense or antisense digoxigenin-UTP-labeled RNA probes were generated by reverse-transcribing the 238-bp progranulin cDNA fragment using either T7 (sense) or T3 (antisense) RNA polymerase after digestion with either Xho (sense) or Xba (antisense). The reverse-transcription was performed using a commercially available Dig-UTP labeling and detection kit (BoehringerMannheim; Indianapolis, IN) according to the manufacturer's instructions. The probe was checked by performing Northern blotting hybridization with mouse kidney total RNA. In brief, two identical membranes containing 15 µg mouse kidney total RNA were hybridized to 100 ng/ml sense or antisense probe and washed. Signals on the membranes were detected as described (Genius System User's guide for membrane hybridization, version 3.0; BoehringerMannheim).
In Situ Hybridization
Paraffin-embedded sections were deparaffinized in xylene (Fisher; Montreal, PQ, Canada) for 4 min, then dehydrated in 100% ethanol followed by progressive hydration in 95%, 70%, and 50% ethanol. The sections were then postfixed in prechilled 4% paraformaldehyde (Fisher), pH 7.4, for 10 min, and washed in 0.5 x SSC for 5 min. Permeabilization of the tissues was achieved by incubating the sections with 3.5 µg/ml proteinase K in 100 mM Tris-HCl, 50 mM EDTA, pH 8.0, at 37C for 15 min. The slides were fixed again in 4% paraformaldehyde for 10 min and rinsed thoroughly in PBS (6 min) and 0.5 x SSC (10 min). The slides were then pre-hybridized in hybridization solution [5 x SSC, 5 x Denhardt's solution, 50% deionized formamide (Fisher), and 250 µg/ml tRNA] at 42C. Three hours later the sections were cleaned with a lint-free tissue and hybridized with 75 ng Dig-UTP labeled progranulin RNA probe in hybridization solution for 18 hr at 42C. After washing, the slides were incubated with conjugated Dig antibody (BoehringerMannheim) and the reaction products were visualized according to the manufacturer's instructions. All experiments were conducted using parallel antisense and sense probes, and in most cases no nonspecific hybridization was observed (this is shown only for the testis sections; see Fig 3). Any experiments showing significant nonspecific hybridization with the sense probe were discarded. Sections from two species, rat and mouse, were analyzed. The numbers of different animals used for each tissue were as follows: testis and epididymis; n = 4; female reproductive organs; n = 3; mammary gland; n = 4; skin; n = 4; gastrointestinal tract; n = 5; urinary system; n = 5; lung; n = 5; liver; n = 4; skeletal muscle; n = 4; heart; n = 5; spleen; n = 5; brain; n = 3. Each tissue was serially sectioned and at least three sections per sample were examined. The species shown in the figure is identified in the legends.
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Purification of the Recombinant Progranulin
Recombinant progranulin was produced as described previously (
Cell Growth Assay
To compare the effect of progranulin on selected cell lines, 1.5 x 104 cells were seeded in 24-well plates (Corning Costar; Cambridge, MA). After 24 hr the medium was replaced with serum-free DMEM for 24 hr. Before addition of the recombinant progranulin, the cells were washed twice in 1 x PBS (pH 7.4) and then incubated with increasing concentrations of recombinant progranulin for 2 days. The cells were trypsinized and counted in a hemocytometer.
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Results |
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Northern Blot Analysis
Total RNAs from several cell lines of various origins were probed for the expression of the progranulin gene (Fig 1A). All epithelial cell lines except the hepatoma line HepG2 expressed the progranulin transcript at high levels. Progranulin transcripts were also abundant in primary cultures of human bronchiolar epithelia and normal human keratinocytes. Myeloid- and lymphoid-derived cell lines also expressed the progranulin transcript, as did leukocytes from patients with acute and chronic myelogenous leukemia. Progranulin transcripts were detected in two leiomyosarcoma-derived lines (SK-UT-1 and SK-LMS-1) but not in undifferentiated L6 rat myoblasts. Neither of the neuron-derived cells lines (PC-12 and SK-N-DZ) expressed progranulin mRNA (not shown). The antisense riboprobe used for in situ hybridization is specific for progranulin transcripts because on Northern blotting analysis of murine kidney mRNA it hybridized to only one mRNA, which had a size of 2.4 kb as predicted for the rodent progranulin transcript (Fig 1B). No hybridization was observed with the sense riboprobe.
Normal Tissue Distribution
Testis and Epididymis.
In the seminiferous tubule, the cells that line the basement membrane (mainly primary spermatogonia) expressed progranulin mRNA, as did the cells between one to three layers deeper within the tubule (Fig 2A). More mature spermatocytes showed little or no expression of progranulin mRNA. In some tubules, Sertoli cells also expressed the progranulin gene. Leydig cells and other cells in the interstitium rarely expressed progranulin. No vascular elements in the testis expressed progranulin. The sense control probe detected no hybridization (Fig 2C). The epididymis showed intense expression of the progranulin gene, which was confined to the epithelial layer (Fig 2B).
Female Reproductive Organs. A section of uterus in the proliferative phase is shown. Glandular epithelium showed pronounced expression of the progranulin gene (Fig 2D). Stromal cells in the proliferative zone expressed the progranulin gene. There was a marked boundary in the basal endometrium which exhibited much lower levels of progranulin hybridization. The smooth muscle cells of the myometrium expressed elevated levels of progranulin mRNA. In the oviducts, only the epithelia lining the oviduct expressed the progranulin mRNA (Fig 2E). The oviductal smooth muscle and vasculature did not express the progranulin gene. In the ovary, hybridization for the progranulin riboprobes was highest in follicular epithelial cells. Hybridization was weak in the corpus luteum and negligible in thecal cells. Progranulin transcripts were also present in oocytes (Fig 2F).
Mammary Gland. Hybridization of the progranulin riboprobe in virginal rat mammary gland was detected exclusively in the glandular epithelium (Fig 2G).
Skin. Epidermal keratinocytes stained strongly for granulin mRNA (Fig 3A). The outer hair follicle hybridized with the progranulin antisense riboprobes (Fig 3B). Cross-sections of the hair follicle showed that the dermal papillae did not express progranulin mRNA. All the epithelial cells at the hair root strongly expressed the progranulin gene. The progranulin gene was expressed in exocrine glands (Fig 3C), although the staining was less intense than that seen in the keratinocytes. Apart from follicles and glandular structures, little if any granulin mRNA was detected in dermal layer fibroblasts or in blood vessels.
Gastrointestinal Tract. Progranulin mRNA appeared to be predominantly located in epithelial cells in the gastrointestinal tract. Squamous epithelium in the esophagus and esophageal/gastric junction stained strongly for progranulin mRNA (Fig 3D). In the small intestine, progranulin was expressed by enterocytes in the deep crypts, with the mRNA levels progressively fading higher in the villus (Fig 3E). A similar pattern was observed in the colon (not shown). Smooth muscle and vasculature showed no progranulin mRNA. However, neuronal ganglia were strongly positive for progranulin mRNA (Fig 3E and insert, white arrow). Lymphocytes in the gut-associated lymphoid tissue hybridized strongly for progranulin mRNA (Fig 3F).
Kidney. Progranulin mRNA was confined to epithelial cells in the kidney. There was weak hybridization in the proximal and distal convoluted tubules of the cortex and in some collecting ducts of the medulla (Fig 3G). No hybridization was observed in the glomerulus or any vascular elements. The strongest hybridization occurred in the transitional epithelium of the ureter, which approached that of skin and intestinal crypt epithelium in intensity.
Lung. Progranulin mRNA was localized in alveolar epithelium only after prolonged incubation with the color reagents, and was confined only to sporadic epithelial cells (Fig 3H). Higher levels of progranulin mRNA were observed in the bronchiolar epithelium (Fig 3I, black arrow), but the intensity of hybridization was considerably lower than in keratinocytes or enterocytes. In the lung, lymphoid cells exhibited the highest levels of progranulin transcript (Fig 3I, open arrow). Progranulin mRNA was absent from cells of the lung vasculature.
Liver. The liver was essentially devoid of cells expressing the progranulin mRNA (not shown).
Skeletal Muscle and Heart. Neither skeletal muscle nor cardiac muscle expressed the progranulin gene (not shown).
Spleen. In the spleen (Fig 4A and Fig 4B), expression of progranulin mRNA occurred predominantly in lymphoid cells on the outer edges of the periarteriolar lymphoid sheaths (white pulp). Cells in the red pulp stained sporadically.
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Brain. Progranulin transcripts were prominent in neurons within the superficial lamina of the cerebral cortex (Fig 5A). Expression was high in the Purkinje cells of the cerebellum (Fig 5B), and in scattered cells in the molecular layer, but less strongly. Expression was absent from granule cells of the internal granular layer. Neurons of the cerebellar roof nuclei showed intense hybridization with the progranulin probe, which was comparable to that of the Purkinje cells (not shown). Progranulin gene expression was high in the hippocampus and was localized to the pyramidal cells and the granule cells. In the posterior portion of the hippocampus, all the granule cells stained evenly (not shown). However, in the mid-hippocampus (Fig 5C and Fig 5D), the granule cells stained more intensely in the Ammon's horn (black arrow) than those in the dentate gyrus (open arrow). Nonneuronal components of the brain, such as ependymal cells and glia, did not express progranulin transcripts.
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Cell Proliferation
Eight cell lines were selected and tested for their proliferative response to progranulin (Fig 6). Two epithelial lines previously shown to respond to progranulin (
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Discussion |
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Progranulin gene products promote tumorigenesis in experimental models (
The results reported here support an important role for progranulin gene expression in both male and female reproductive tissues. In the adult testes, the highest levels of progranulin mRNA are associated with immature spermatocytes and spermatogonia but not with more mature spermatozoa (Fig 2A). This is presumably correlated with the synthesis of acrogranin, a high molecular weight progranulin product found within acrosomes (
Progranulin is a potent mitogen for fibroblasts (
The peripheral immune system shows very strong expression of the progranulin gene in lymphoid tissue of the lung, gut, and spleen. The distribution of progranulin mRNA in immune tissues does not correspond to the expected patterns of immune cell proliferation, implying a nonmitogenic function for progranulin products in the immune system. In the spleen, the progranulin transcript is confined mainly to marginal cells of the periarteriolar lymphoid sheath (PALS) (Fig 4A and Fig 4B). These cells are largely T-lymphocytes. The B-lymphocytic population within the interior of the PALS shows less extensive progranulin staining. Lymphoid aggregates in the lung and gut express progranulin mRNA at high levels (Fig 3I and Fig 3F). Cell lines derived from both B- and T-lymphomas express the progranulin gene in culture (Fig 1A). The cellular specificity of progranulin expression in the peripheral immune system, which is particularly apparent in the spleen, reflects stringent regulation of the progranulin gene in the immune system. Granulins were initially detected in activated inflammatory cells (
In earlier studies we reported that the levels of progranulin mRNA in the brain were very low compared to those in other tissues ( (
In conclusion, progranulin gene expression is closely associated with epithelial cells in somatic tissues. Progranulin gene expression is prominent in rapidly self-renewing epithelia, such as the skin and gut. In the gut, the expression is greatest in the highly proliferative cells of the deep crypts and becomes negligible in the terminally differentiated cells higher in the villus. Chronically nonproliferating highly differentiated epithelia, such as the lung alveolae and kidney tubules, show much lower levels of progranulin expression, although immortalized or neoplastic cell lines from low progranulin-expressing tissues, such as the kidney (MDCK) or the lung (A549), express the gene highly and respond to the recombinant protein. This is consistent with results showing that overexpression of progranulin in some epithelial cell lines leads to a more proliferative phenotype (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported in part by MRC of Canada grant MT11288 (AB) and NIH grant CA71023 (JH). AB is a Senior Chercheur Boursier of the Fonds de la Recherche en Santé du Québec. ZH is the recipient of a studentship from the Research Institute of the Royal Victoria Hospital, Montréal.
Received for publication February 22, 2000; accepted February 23, 2000.
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