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Volume 52 (2): 153-156, 2004
Copyright ©The Histochemical Society, Inc.
RAPID COMMUNICATION |
Laminins 
2 and 4 in Pancreatic Acinar Basement Membranes Are Required for Basal Receptor Localization
Department of Internal Medicine, Renal Division, Washington University School of Medicine, St Louis, Missouri (JHM,CL), and CROET, Oregon Health Sciences University, Portland, Oregon (BLP)
Correspondence to: Jeffrey H. Miner, Renal Division, Box 8126, Washington U. School of Medicine, 660 South Euclid Ave., St Louis, MO 63110. E-mail: minerj{at}wustl.edu
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Summary |
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 Top Summary Literature Cited |
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Basement membranes (BMs) are thin layers of extracellular matrix (ECM) found at the basal surface of many cell types, including epithelial cells. BMs present growth, differentiation, and anti-apoptotic signals and provide structural support to cells, compartmentalize tissues, and serve as filters. The structure and function of BMs depend on their complement of laminins, a family of
ß
heterotrimeric glycoproteins. We found that laminins containing the 2 and 4 chains are the major laminins in pancreatic acinar BMs. Importantly, these laminins were required for proper basal localization on acinar cells of two laminin receptors, dystroglycan and integrin 6ß4. (J Histochem Cytochem 52:153–156, 2004)
Key Words: laminin • integrin • pancreas • merosin • dystroglycan
ALL BASEMENT MEMBRANES (BMs) contain laminin, a family of five -, four ß-, and three -chains that assemble to form at least 15 different ß heterotrimers, designated laminins-1 to -15 (Colognato and Yurchenco 2000
). Laminin mutations cause defects in humans and mice, including amyelination and muscular dystrophy from loss of the 2-chain and skin blistering from loss of laminin-5 (3ß32). Knockout of the mouse Lama4, Lama5, Lamb2, and Lamc1 genes have revealed roles for 4 in the vasculature and in neuromuscular junction organization; for 5 in brain, limb, placenta, kidney, and lung development, for ß2 in neuromuscular and glomerular function, and for 1 in endoderm differentiation (Colognato and Yurchenco 2000; Patton et al. 2001; Nguyen et al. 2002; Thyboll et al. 2002). These defects correlate with the normal patterns of deposition of the affected chains, demonstrating the importance of an understanding of the distribution of laminins in BMs.
Here we focus on the distribution of laminins in the pancreas. The exocrine pancreas contains acinar cells, epithelia that secrete digestive enzymes. The endocrine pancreas consists of islets of Langerhans, which contain insulin-secreting ß-cells and capillaries for delivering insulin to the bloodstream. Many studies in vitro suggest that laminins are involved in pancreatic cell function. To interpret and extend these findings in vivo, it is important to know the distribution of laminins in the pancreas. A recent study concluded that acinar tissue contains laminin-10 (5ß11) but not laminin-2 (2ß11) (Jiang et al. 2002). In contrast, we show here that acinar BMs have little or no laminin 5 but are rich in laminins 2 and 4.
The primary antibodies used are listed in Table 1. Secondary antibodies were obtained from Chemicon (Temecula, CA). Fresh pancreases were flash-frozen, sectioned, fixed, and stained as described (Patton et al. 2001). Control mice had mixed 129-C57BL/6J backgrounds; similar results were obtained in B6CBAF1 and outbred ICR mice. Lama2 (dy2J and dy3k) and Lama4 mutant mice have been previously described (Xu et al. 1994; Miyagoe et al. 1997; Patton et al. 2001).
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Acinar cell BMs in normal pancreas were labeled uniformly by antibodies to laminins
2 and 4 (Figures 1A and 1B)
. In contrast, laminin 5 was undetectable in acinar BMs despite intense staining in blood vessels and capillaries (Figure 1C). Laminins ß1 and ß2 were both present throughout the acinar cell BM (Figures 2A and 2B)
; laminin 1 was ubiquitous in pancreatic BMs (data not shown), as reported (Jiang et al. 2002).
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Laminin
2 was previously reported to be present only in pancreatic blood vessels (Jiang et al. 2002), using MAb 4H8-2 against the NH2-terminal 2 short arm. We found 2 in acinar cell BMs using both 4H8-2 (Figure 1A) and an antiserum directed against the COOH-terminal G-domains (data not shown). Neither antibody stained pancreatic or other tissue from 2-null mutant mice (Lama2dy-3k; Figure 1F, and data not shown), demonstrating their specificity for the 2-chain. Anti-4 stained 2-deficient acinar BMs weakly (Figure 1G).
To ask if laminins containing 2 and 4 were required for acinar BM formation, we examined mice bearing both 2 and 4 mutations. First, we assessed double-mutant mice harboring a weak Lama2 mutation (Lama2dy-2J) and a Lama4 null mutation. The Lama2dy-2J mutation truncates the 2-chain short arm, prevents polymerization of 2dy2J-containing trimers, and disrupts BM assembly in muscle and nerve (Colognato and Yurchenco 2000). Both antibodies to 2 stained acinar BMs from adult homozygous Lama2dy-2J, Lama4 -/- mutant mice in a weak and interrupted fashion (Figure 1K, and data not shown); 4 was undetectable (Figure 1L), confirming specificity of the 4 antibody. The effect of combined 2–4 deficiency on overall BM composition was drastic: neither nidogen-1 nor collagen IV was concentrated in a normal linear pattern in acinar BMs (Figures 1N and 1O).
Second, we assessed BM composition in mice homozygous for null mutations in both Lama2 and Lama4, which die before 2 weeks of age (BLP, unpublished data). Pancreases from 12-day-old doubly null mice lacked 2 and 4 immunoreactivity (Figures 1U and 1V), and there was no compensation by other -chains (data not shown). Moreover, little nidogen-1 or collagen IV accumulated, except in vessels (Figures 1X and 1Y). Therefore, the very existence of acinar BMs in 2;4 double-null mice is suspect, and H&E staining revealed that acinar cell polarization was impaired (data not shown).
Finally, we asked how changes in laminin composition affected localization of laminin receptors. In normal pancreas, we detected ß1-class integrins only in the vasculature (Figure 2E). In contrast, dystroglycan (Figure 2C) and integrin 6ß4 (Figures 2F and 2G) were concentrated on acinar cell basal surfaces. Basal localization of dystroglycan was lost in the absence of laminin 2 (Figure 2D), consistent with their direct interaction (Colognato and Yurchenco 2000). Interestingly, the basal localization of integrin 6ß4 was maintained in the same cells (Figures 2H and 2I) but was significantly reduced in the absence of both 2- and 4-laminins, compared to controls (Figures 2F–2O). The results suggest selective interactions between laminin 2 and dystroglycan and between laminin 4 and integrin 6ß4.
We conclude that laminin-2 (2ß11) and laminin-4 (2ß21), containing the 2-chain, are the major laminins of acinar cell BMs in adult mice. Additional isoforms include laminins-8 (4ß11) and/or -9 (4ß21), containing the 4-chain. However, there is little or no laminin-10 in acinar BMs, previous data notwithstanding (Jiang et al. 2002). We also suggest that dystroglycan and integrin 6ß4 are receptors for distinct laminins on acinar cells and that their basal localization is organized by BM composition. The role of cell/matrix interactions in pancreas structure and function remains to be determined, but defects in acinar cell polarization (data not shown) in the apparent absence of basement membrane suggest that they are critical.
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Acknowledgments |
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Supported by grant R01 GM060432 from the NIH and in part by a research grant from the March of Dimes to JHM, and by NIH RO1 NS40759 and a grant from the Muscular Dystrophy Association to BLP.
We are grateful to those investigators who provided antibodies used in this study. We are especially grateful to Drs Yuko Miyagoe–Suzuki, Shin'ichi Takeda, and Karl Tryggvason for providing the 2-null and 4-null mice.
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Footnotes |
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Received for publication July 23, 2003; accepted October 23, 2003
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Literature Cited |
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TopSummaryLiterature Cited |
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Abrahamson DR, Irwin MH, St John PL, Perry EW, Accavitti MA, Heck LW, Couchman JR (1989) Selective immunoreactivities of kidney basement membranes to monoclonal antibodies against laminin: localization of the end of the long arm and the short arms to discrete microdomains. J Cell Biol 109:3477–3491
Cheng Y-S, Champliaud M-F, Burgeson RE, Marinkovich MP, Yurchenco PD (1997) Self-assembly of laminin isoforms. J Biol Chem 272:31525–31532
Colognato H, Yurchenco PD (2000) Form and function: the laminin family of heterotrimers. Dev Dyn 218:213–234[Medline]
Ervasti JM, Campbell KP (1993) A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 122:809–823
Jiang FX, Naselli G, Harrison LC (2002) Distinct distribution of laminin and its integrin receptors in the pancreas. J Histochem Cytochem 50:1625–1632
Miyagoe Y, Hanaoka K, Nonaka I, Hayasaka M, Nabeshima Y, Arahata K, Takeda S (1997) Laminin alpha2 chain-null mutant mice by targeted disruption of the Lama2 gene: a new model of merosin (laminin 2)-deficient congenital muscular dystrophy. FEBS Lett 415:33–39[Medline]
Nguyen NM, Miner JH, Pierce RA, Senior RM (2002) Laminin alpha5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung. Dev Biol 246:231–244[Medline]
Patton BL, Cunningham JM, Thyboll J, Kortesmaa J, Westerblad H, Edstrom L, Tryggvason K, et al. (2001) Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4. Nature Neurosci 4:597–604[Medline]
Sasaki T, Mann K, Miner JH, Miosge N, Timpl R (2002) Domain IV of mouse laminin ß1 and ß2 chains: structure, glycosaminoglycan modification and immunochemical analysis of tissue contents. Eur J Biochem 269:431–442[Medline]
Talts JF, Sasaki T, Miosge N, Gohring W, Mann K, Mayne R (2000) Structural and functional analysis of the recombinant G domain of the laminin 4 chain and its proteolytic processing in tissues. J Biol Chem 275:35192–35199
Thyboll J, Kortesmaa J, Cao R, Soininen R, Wang L, Iivanainen A, Sorokin L, et al. (2002) Deletion of the laminin alpha4 chain leads to impaired microvessel maturation. Mol Cell Biol 22:1194–1202
Xu H, Wu X-R, Wewer UM, Engvall E (1994) Murine muscular dystrophy caused by a mutation in the laminin 2 (Lama2) gene. Nature Genet 8:297–302[Medline]
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