(743.16) Loss of Myosin Vb leads to dysregulation of colonic goblet cell structure and function

Poster Board Number: E343

Jessica Digrazia (Medical University of South Carolina), Melinda Engevik (Medical University of South Carolina, Medical University of South Carolina), Amy Engevik (Medical University of South Carolina)

Background: Intestinal goblet cells secrete a protective mucus layer preventing bacteria and harmful ingested substances from contacting the columnar epithelial cells that line the intestine. Single cell RNAseq data has revealed that goblet cells have high expression of Myosin 5b (Myo5b), a molecular motor that regulates vesicle trafficking in epithelial cells. Myo5b is most widely studied in the small intestine, but it’s role in the colonic epithelium and particularly goblet cells is unknown. A recent study found that Myo5b expression was reduced in inflammatory bowel disease (IBD), a condition that is characterized by dysfunctional mucus; potentially providing a link between Myo5b and goblet cell function. We hypothesized that loss of Myo5b would perturb goblet cell mucus production.

Methods: Adult control and intestinal specific tamoxifen inducible VillinCreERT2;Myo5bflox/flox (Myo5b KO) mice were injected intraperitoneally with 2 mg of tamoxifen to deplete intestinal Myo5b. After four days colonic tissue was collected and analyzed.

Results: We found that loss of Myo5b in vivo results in reduced number of goblet cells in the colon by Hamp;E staining. Muc2 immunostaining and alcian blue staining similarly demonstrated a reduction of mucin within goblet cells. Mucin proteins are heavily o-glycosylated which makes up 80% of the molecular weight. Normally, mucin structures are terminated by sialic acid which provides a negative charge that protects the mucus protein from bacterial degradation. Staining for sialic acid with the lectin SNA revealed a decrease in sialic acid levels in the mucus of Myo5b KO mice compared to controls. Once sialic acid is removed from mucins, the underlying glycan structures are exposed. Using the lectin WGA to identify the internal glycan oligosaccharide n-acetyl glucosamine, we observed increased exposure of n-acetyl glucosamine in Myo5b KO mice compared to control mice; which was consistent with our sialic acid findings. Intestinal goblet cells also secrete wound healing factors including the peptide Trefoil factor 3 (TFF3). Similar to the pattern we observed for Muc2 and sialic acid content, we found decreased levels of TFF3 in the mucosa of Myo5b KO mice.

Conclusions: Our data shows that loss of functional Myo5b in the colonic epithelium results in decreased goblet cell numbers and alterations in mucin composition. These data suggest that Myo5b may play an important role in goblet cell mucus production and that mutations affecting Myo5b function may contribute to IBD.





Figure 1: Adult intestinal specific tamoxifen inducible Myo5b KO mice exhibit changes in colonic goblet cells. (A & C) Hemat­oxylin & Eosin staining shows altered colonic architecture with a decrease in the number of goblet cells in the colon of Myo5b KO mice compared to control mice. (B) Alcian blue staining identifying acidic mucins in goblet cells demonstrates changes in goblet cell structure and mucin density. (C) Quantification of goblet cells per crypt in control and Myo5b KO mice *p < 0.05.; Figure 2: Fluorescence imaging of mucin proteins and glycosylation reveals alterations in goblet cell mucus composition in Myo5b KO colon. Immunostaining for MUC2 in green (A) and TFF3 in purple (B) shows decreased MUC2 and TFF3 intensity in Myo5b KO mice compared to control mice. Lectin staining for sialic acid (SNA in yellow) (C) and n-acetyl glucosamine (using WGA in pink) (D) revealed altered glycan structures in the colons of Myo5b KO mice. n=4-6 mice per group.