Laminins α2 and α4 in Pancreatic Acinar Basement Membranes Are Required for Basal Receptor Localization

Abstract

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 αβγ heterotrimeric glycoproteins. We found that laminins containing the α2 and α4 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 α6β4.

All basement membranes (BMs) contain laminin, a family of five α-, four β-, and three γ-chains that assemble to form at least 15 different αβγ 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 α2-chain and skin blistering from loss of laminin-5 (α3β3γ2). Knockout of the mouse Lama4, Lama5, Lamb2, and Lamc1 genes have revealed roles for α4 in the vasculature and in neuromuscular junction organization; for α5 in brain, limb, placenta, kidney, and lung development, for β2 in neuromuscular and glomerular function, and for γ1 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 β-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 (α5β1γ1) but not laminin-2 (α2β1γ1) (Jiang et al. 2002). In contrast, we show here that acinar BMs have little or no laminin α5 but are rich in laminins α2 and α4.

Table 1

Primary antibodies

Antibody to	Clone	Host/Antigen Species	Source
Laminin α2	4H8–2	Rat/human	Alexis Biochemicals, Montreal, PQ, Canada
	–	Rabbit/human	Dr. P.D. Yurchenco (Cheng et al. 1997)
Laminin α4	–	Rabbit/mouse	Dr. R. Timpl (Talts et al. 2000)
Laminin α5	–	Rabbit/mouse	Miner Laboratory (Nguyen et al. 2002)
Laminin β1	5A2	Rat/mouse	Dr. D.R. Abrahamson (Abrahamson et al. 1989)
Laminin β2	–	Rabbit/mouse	Dr. R. Timpl (Sasaki et al. 2002)
Laminin γ1	A5	Rat/mouse	Chemicon, Temecula, CA
Nidogen-1	ELM7	Rat/mouse	Chemicon
Collagen IV	−	Goat/human	Southern Biotechnology Associates, Birmingham, AL
Dystroglycan	IIH6	Mouse/rabbit	Dr. K.P. Campbell (Ervasti and Campbell 1993)
Integrin α6	GoH3	Rat/human	Chemicon
Integrin β1	MB1.2	Rat/mouse	Chemicon
Integrin β4	346–11A	Rat/mouse	BD Pharmingen, San Diego, CA

Figure 1

(A-Y) Distribution of laminin α-chains, nidogen-1, and collagen IV in control and mutant pancreases, as indicated. (A-O) seven-week-old mice; (P-Y) 12-day-old mice. Inset in B shows a high-power view of acinar BMs (arrowheads). i, islet of Langerhans. Bar = 50 μm.

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 (dy^2J and dy^3k) and Lama4 mutant mice have been previously described (Xu et al. 1994; Miyagoe et al. 1997; Patton et al. 2001).

Acinar cell BMs in normal pancreas were labeled uniformly by antibodies to laminins α2 and α4 (Figures 1A and 1B). In contrast, laminin α5 was undetectable in acinar BMs despite intense staining in blood vessels and capillaries (Figure 1C). Laminins β1 and β2 were both present throughout the acinar cell BM (Figures 2A and 2B); laminin γ1 was ubiquitous in pancreatic BMs (data not shown), as reported (Jiang et al. 2002).

Figure 2

(A-O) Distribution of laminin β-chains, dystroglycan, and integrins in control and mutant pancreases, as indicated. (A-K) seven-week-old mice; (L-O) 12-day-old mice. Bar: A-K = 50 μm; L-O = 25 μm.

Laminin α2 was previously reported to be present only in pancreatic blood vessels (Jiang et al. 2002), using MAb 4H8–2 against the NH₂-terminal α2 short arm. We found α2 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 α2-null mutant mice (Lama2^dy-3k; Figure 1F, and data not shown), demonstrating their specificity for the α2-chain. Anti-α4 stained α2-deficient acinar BMs weakly (Figure 1G).

To ask if laminins containing α2 and α4 were required for acinar BM formation, we examined mice bearing both α2 and α4 mutations. First, we assessed double-mutant mice harboring a weak Lama2 mutation (Lama2^dy-2J) and a Lama4 null mutation. The Lama2^dy-2J mutation truncates the α2-chain short arm, prevents polymerization of α2^dy2J-containing trimers, and disrupts BM assembly in muscle and nerve (Colognato and Yurchenco 2000). Both antibodies to α2 stained acinar BMs from adult homozygous Lama2^dy-2J, Lama4 −/− mutant mice in a weak and interrupted fashion (Figure 1K, and data not shown); α4 was undetectable (Figure 1L), confirming specificity of the α4 antibody. The effect of combined α2–α4 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 α2 and α4 immunoreactivity (Figures 1U and 1V), and there was no compensation by other α-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 α2;α4 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 β1-class integrins only in the vasculature (Figure 2E). In contrast, dystroglycan (Figure 2C) and integrin α6β4 (Figures 2F and 2G) were concentrated on acinar cell basal surfaces. Basal localization of dystroglycan was lost in the absence of laminin α2 (Figure 2D), consistent with their direct interaction (Colognato and Yurchenco 2000). Interestingly, the basal localization of integrin α6β4 was maintained in the same cells (Figures 2H and 2I) but was significantly reduced in the absence of both α2-and α4-laminins, compared to controls (Figures 2F-2O). The results suggest selective interactions between laminin α2 and dystroglycan and between laminin α4 and integrin α6β4.

We conclude that laminin-2 (α2β1γ1) and laminin-4 (α2β2γ1), containing the α2-chain, are the major laminins of acinar cell BMs in adult mice. Additional isoforms include laminins-8 (α4β1γ1) and/or -9 (α4β2γ1), containing the α4-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 α6β4 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.