Effects of Muclin (Dmbt1) deficiency on the gastrointestinal system

Abstract

The Dmbt1 gene encodes alternatively spliced glycoproteins that are either membrane-associated or secreted epithelial products. Functions proposed for Dmbt1 include it being a tumor suppressor, having roles in innate immune defense and inflammation, and being a Golgi-sorting receptor in the exocrine pancreas. The heavily sulfated membrane glycoprotein mucin-like glycoprotein (Muclin) is a Dmbt1 product that is strongly expressed in organs of the gastrointestinal (GI) system. To explore Muclin's functions in the GI system, the Dmbt1 gene was targeted to produce Muclin-deficient mice. Muclin-deficient mice have normal body weight gain and are fertile. The Muclin-deficient mice did not develop GI tumors, even when crossed with mice lacking the known tumor suppressor p53. When colitis was induced by dextran sulfate sodium, there was no significant difference in disease severity in Muclin-deficient mice. Also, when acute pancreatitis was induced with supraphysiological caerulein, there was no difference in disease severity in the Muclin-deficient mice. Exocrine pancreatic function was impaired, as measured by attenuated neurohormonal-stimulated amylase release from Muclin-deficient acinar cells. Also, by [(35)S]Met/Cys pulse-chase analysis, traffic of newly synthesized protein to the stimulus-releasable pool was significantly retarded in Muclin-deficient cells compared with wild type. Thus Muclin deficiency impairs trafficking of regulated proteins to a stimulus-releasable pool in the exocrine pancreas.

Fig. 1. Targeting strategy and production of Muclin-deficient mice

(A) Schematic diagram of WT gene in the target region, the targeting construct, and the targeted gene. (B) Southern blots of Muclin WT (+/+), heterozygous (+/−), and knockout (−/−) genomic DNA: digested with XbaI and hybridized with the 5’-outside probe to verify correct targeting; digested with BamHI or EcoRI and hybridized with a GFP probe to demonstrate there was a single integration site and there were no nonspecific transgene insertions.

Fig. 2. Analysis of Muclin protein expression in WT (+/+) and Muclin-deficient (−/−) mice

(A) Western blot for Muclin in pancreas (Pan) and small intestine (Int). There is no signal in samples from the knockout mice. (B) Metabolic [³⁵S]sulfate incorporation into pancreatic acinar cell proteins. The high molecular mass Muclin band shows [³⁵S] incorporation in WT cells but not in the knockout cells. Other sulfated species are unchanged in the knockout. (C) Coomassie blue stained gels showing identical pancreas and isolated zymogen granule protein compositions in WT (+/+) and Muclin-deficient (−/−) mice. Homog = homogenate of total pancreas; ZG = isolated zymogen granules. (D) Muclin immunohistochemistry in WT and Muclin-deficient pancreas and small intestine. (i) In the WT pancreas the zymogen granules are strongly labeled and (ii) there is no labeling in the knockout tissue. (iii) In the WT small intestine labeling is mostly confined to the crypt epithelium above the Paneth cells and (iv) there is no labeling in the knockout tissue.

Fig. 3. Dextran sulfate sodium-induced colitis in WT and Muclin-deficient mice

(A) Body weight loss during DSS administration. Mice began to lose body weight by 5 days of treatment, and there were no significant differences comparing Muclin-deficient (KO) to WT mice. (B) Hemoccult scores during DSS administration. There were no significant differences comparing Muclin-deficient to WT mice. (C) Colon length in controls and after 7 days DSS administration. There were no significant differences in controls comparing Muclin-deficient to WT mice. After 7 days DSS administration both WT and Muclin-deficient mice had significantly shorter colons as compared to untreated mice (p=0.0002) and there was not a significant difference comparing Muclin-deficient to WT mice. (D) Histopathology scores of WT and Muclin-deficient colon after 7 days DSS treatment. All mice showed pathological changes but there were no significant differences comparing DSS-treated Muclin-deficient and WT mice. Data are means ± SE from 5 WT and 5 Muclin-deficient mice.

Fig. 4. Histology and Muclin immunohistochemistry in dextran sulfate sodium-induced colitis in WT mice

(A and C) Routine hematoxylin and eosin (H&E) histology. (B and D) Immunohistochemistry for Muclin (Muclin IHC). The control colon shows (A) normal histology and (B) Muclin immunoreactivity is mostly in the surface epithelium with little labeling deeper in the crypts. After 7 days DSS treatment, (C) the epithelium is focally destroyed and there are inflammatory infiltrates; (D) Muclin immunoreactivity is more intense and cells deep in the crypts now express immunoreactive Muclin. Representative images from 5 each Control and DSS-treated WT mice.

Fig. 5. Biochemical analysis of caerulein-induced pancreatitis in Muclin-deficient mice as compared to WT

Mice were injected 7 times at hourly intervals with 50 µg/kg caerulein i.p. (A) At sacrifice trunk blood was collected for measurement of serum amylase. (B) The pancreas was harvested and wet weight was recorded followed by lyophilization to dryness to determine tissue water content. (C) The tissue was used to measure myeloperoxidase (MPO) activity as a measure of neutrophil infiltration. Data are means ± SE from 4 individual animals per time point and genotype. There were no significant differences in the severity or resolution of pancreatitis comparing Muclin-deficient (KO) to WT mice.

Fig. 6. Histology of caerulein-induced pancreatitis in Muclin-deficient mice as compared to WT

(A) WT and (B) Muclin-deficient untreated (Control) pancreas. There were no apparent differences comparing Muclin-deficient to WT pancreas. (C) WT and (D) Muclin-deficient pancreas 7 hr after starting supraphysiological caerulein injections. Both genotypes exhibited similar degrees of edema, inflammatory cell infiltrates, and acinar cell apoptosis. (E) WT and (F) Muclin-deficient pancreas 7 days after caerulein treatment. There were no histological differences comparing Muclin-deficient to WT mice. (I) = islet of Langerhans. Representative images from 4 each WT and Muclin-deficient mice for each time point.

Fig. 7. Amylase release from Muclin-deficient and WT pancreatic acini in response to cholinergic (carbachol) or caerulein stimulation

Acini were isolated and incubated with the indicated concentrations of stimulus for 30 min at 37°C. Amylase released into the media is expressed as percent of the initial cellular content corrected for activity in the media at the start of the release period. (A) Carbachol stimulated amylase release. There were no significant differences in amylase release comparing Muclin-deficient (KO) to WT acini. (B) Caerulein stimulated amylase release. (*) p<0.05 comparing WT to Muclin-deficient (KO) cells. Data are means ± SE from acinar preparations from 5 mice each per genotype.

Fig. 8. [³⁵S]met/cys pulse-chase analysis of protein trafficking through the secretory pathway in WT and Muclin-deficient pancreatic acini

Pancreatic acini were prepared and pulse-labeled with [³⁵S]met/cys, washed, and then chased for the indicated times. Where indicated, cells were stimulated with 1 µM carbachol and 1 mM 8-Br-cAMP for 0.5 hr. [³⁵S]amylase in the media was quantified from the phosphor storage data and are expressed as percent of labeled amylase in the media relative to the total in the cell pellets at the end of the labeling period. The data are from 6 each WT and Muclin-deficient (KO) acinar preparations. Basal secretion (unstimulated) is indicated by the dashed lines. Stimulated secretion is shown in solid lines. (*) Stimulated secretion between 0.5−1 hr and between 1−1.5 hr of chase was significantly less in Muclin-deficient acini (p=0.029, 0.0031, respectively).