Pancreatic LKB1 deletion leads to acinar polarity defects and cystic neoplasms - PubMed (original) (raw)

Pancreatic LKB1 deletion leads to acinar polarity defects and cystic neoplasms

Aram F Hezel et al. Mol Cell Biol. 2008 Apr.

Abstract

LKB1 is a key regulator of energy homeostasis through the activation of AMP-activated protein kinase (AMPK) and is functionally linked to vascular development, cell polarity, and tumor suppression. In humans, germ line LKB1 loss-of-function mutations cause Peutz-Jeghers syndrome (PJS), which is characterized by a predisposition to gastrointestinal neoplasms marked by a high risk of pancreatic cancer. To explore the developmental and physiological functions of Lkb1 in vivo, we examined the impact of conditional Lkb1 deletion in the pancreatic epithelium of the mouse. The Lkb1-deficient pancreas, although grossly normal at birth, demonstrates a defective acinar cell polarity, an abnormal cytoskeletal organization, a loss of tight junctions, and an inactivation of the AMPK/MARK/SAD family kinases. Rapid and progressive postnatal acinar cell degeneration and acinar-to-ductal metaplasia occur, culminating in marked pancreatic insufficiency and the development of pancreatic serous cystadenomas, a tumor type associated with PJS. Lkb1 deficiency also impacts the pancreas endocrine compartment, characterized by smaller and scattered islets and transient alterations in glucose control. These genetic studies provide in vivo evidence of a key role for LKB1 in the establishment of epithelial cell polarity that is vital for pancreatic acinar cell function and viability and for the suppression of neoplasia.

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Figures

FIG. 1.

(a) Pdx1-Cre is expressed in common pancreas progenitors at E8 to E12, leading to the deletion of the conditional Lkb1 null allele in all pancreas epithelial cell types, acini, ducts, and islets. (b) Western blotting analysis of Lkb1 in PD1 p-Lkb1 and WT pancreas. (c) Gross and histological appearances of adult WT (i, iii, and v), p-Lkb1 pancreas (ii, iv, and vi); note the spleen adherent to the inferior border of the cystic mass, which involved the entire pancreas. Simple cystic lining epithelium is shown with red arrowhead and inset (iv). p-Lkb1 PD1 pancreases show intact acinar cells and occasional luminal dilatation (panel vi inset, yellow arrow) compared to that of WT (v), as well as apoptotic bodies (panel vi, blue arrow). (d) Loss of acini and replacement with ductal epithelial structures (arrows) and reactive fibrosis (asterisk) are seen at PD2 to PD8 in p-Lkb1 pancreases.

FIG. 2.

Metabolic profiles of p-Lkb1. (a and b) PD1 serum amylase levels are increased (a), and perinatal weight gain is diminished (b) in p-Lkb1 mice. (c) Nonfasting blood glucose levels are slightly elevated following birth and are then comparable by PD8. (d) Six-week-old p-Lkb1 mice demonstrate improved glucose tolerance compared to their littermate controls. IP, intraperitoneal.

FIG. 3.

(a) Immunohistochemistry analysis of PD1 carboxypeptidase, PD3 DBA, and PD3 TUNEL (arrows highlight TUNEL-positive cells in p-Lkb1). (b) Immunofluorescence analysis of PD2 and PD4, demonstrating both acinar and ductal cell contributions to metaplastic lesions. Terminal CK19 staining ducts (arrows in panels i and ii) are seen closely associated with acinar units in the indicated WT specimens; otherwise, acinar cells with cytoplasmic amylase staining do not exhibit membranous CK19 staining. Both amylase (acinar and cytoplasmic) and CK19 (ductal and membranous) marking as seen within rare cells within metaplastic lesions (arrows in panels iii, iv, and v and inset). DAPI, 4′,6′-diamidino-2-phenylindole.

FIG. 4.

(a) Immunohistochemistry demonstrates HES-1 staining in p-Lkb1 PD3 ductal structures and adult cystadenomas (arrows). (b) Western blotting analysis demonstrates increased phosphor-STAT3 and activated carboxypeptidase (arrowhead, middle gel) in the PD1 p-Lkb1 pancreas. (c) TEM of E18.5 p-Lkb1 pancreas demonstrates grossly abnormal acinar units with a loss of basal nuclear location (panel iv, nuclei marked with asterisks), the absence of apically located tight and adherens junctions (panels ii and v, arrows indicate the junction in the WT and the expected location in p-Lkb1) and lateralization of microvilli (panels iii and vi, arrows indicate lateral surface).

FIG. 5.

(a) Apical localization of e-cadherin (e-cad) (panel i, arrows) is absent in p-Lkb1 pancreas; costaining (insets) for amylase and DAPI (4′,6′-diamidino-2-phenylindole) demonstrates full acinar differentiation and an associated loss of basal nuclear localization. Phalloidin marks the apical capping and localization of actin in the WT (panel ii, arrows) that is markedly reduced in p-Lkb1 (v). Tubulin has a punctate and irregular distribution in p-Lkb1 (vi) compared with that of the WT (iii). (b) aPKC is seen laterally displaced from apices in p-Lkb1 PD1 animals (panels iv and v, arrows) as is the TJ marker, ZO1 (vi).

FIG. 6.

Biochemical evaluation of PD1 whole-pancreas lysates. (a to c) Mutant pancreases demonstrated a loss of phosphorylation of AMPK, MARK2 and MARK3, and SAD-B compared with that of the WT, as well as downstream targets ACC and MAP4. While AMPK and MARK2 levels were equivalent in the WT and mutant animals, MARK3 protein levels diminished in mutants. (d) Western blotting analysis of LC3 at PD1 in starved and fed WT and p-LKB1 whole-pancreas lysates. The p-Lkb1 and WT pancreases have similar LC3 ratios under both fed and starved conditions. (e) MLC2 Ser19 phosphorylation remains elevated among p-Lkb1 pancreases and compared with WT controls among PD1 animals starved for 16 h following birth.

FIG. 7.

(a) Immunohistochemistry analysis of Lkb1 in E16.5 and PD8 pancreases. Lkb1 is expressed in both islet and acinar cells at E16.5 but is restricted in adulthood to islets (asterisks mark islets at PD8). Intense staining between acinar units and islets of p-Lkb1 at PD8 represents macrophages, fibroblasts, and infiltrative reactive stroma. (b) Immunofluorescence demonstrates similar distributions and contributions of α, β, and δ cells in E16.5 p-Lkb1 pancreases. (c) Immunohistochemistry analysis of endocrine markers at E16.5 demonstrates the presence of α, β, and δ cells. Abbreviations: ins, insulin; gcg, glucagon; sst, somatostatin.

FIG. 8.

(a) Immunohistochemistry analysis of PD1 pancreases for insulin (ins), glucagon (gcg), and somatostatin (sst) demonstrates scattered endocrine cells among p-Lkb1 pancreases compared with those that are coalesced at islets. (b) Evaluation of PD16 p-Lkb1 pancreases demonstrates smaller islets compared with those of the WT, however, with normal organization and contribution of α, β, and δ cells.

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Pancreatic LKB1 deletion leads to acinar polarity defects and cystic neoplasms - PubMed (original) (raw)