The International Journal of Biochemistry & Cell Biology
Human intestinal MUC17 mucin augments intestinal cell restitution and enhances healing of experimental colitis
Introduction
Mucin-type proteins, categorized as secretory or membrane bound (1), are located at the interface of epithelial surfaces and the environment. These proteins are thought to be integral to the epithelial defense of respiratory, digestive, ocular, and reproductive surfaces. The membrane-bound mucins are characterized by an extracellular region composed of a short amino terminal domain, followed by a large, heavily O-glycosylated tandem repeat domain which accounts in part for their protective function. This large (>4000 amino acids) structural domain is followed by a globular region just proximal to the membrane that is made up of two Cys-rich domains (CRD1 and CRD2), each with similarity to epidermal growth factor (EGF)-like motifs, separated by a linker (L) domain containing a SEA (sea-urchin sperm protein, enterokinase and agrin) module. C-terminal to the CRD1-L-CRD2 unit is a transmembrane segment followed by a small cytoplasmic domain. An overview of this structure of MUC17 is shown schematically in Fig. 1. The related membrane-bound mucin genes MUC3A/B, MUC12, and MUC17 are clustered on chromosome 7q22, and are highly expressed in intestinal tissues at the apical surface of enterocytes. The mouse Muc3 gene (Shekels et al., 1998) is most similar in sequence and chromosomal localization to the human MUC17 gene (Gum et al., 1997, Crawley et al., 1999, Williams et al., 1999).
We have previously shown that GST-tagged recombinant mouse Muc3-CRD1-L-CRD2 (also termed Muc3 EGF1, 2) inhibits cellular apoptosis and accelerates cell migration over surfaces in vitro; and also promotes healing in mouse models of ulcerative colitis (Ho et al., 2006). The Muc3-GST-CRD1-L-CRD2 protein does not directly activate EGF receptors, but the specific mechanisms responsible for the actions of the mucin-derived recombinant proteins have not been determined to date. These findings constitute the rationale for studying the function of endogenous membrane-bound MUC17 mucin and the testing of a human recombinant mucin protein containing a bivalent display of Cys-rich EGF-like CRD domains for potential therapeutic use.
In the present work, we sought to determine: (1) if endogenous human MUC17 demonstrated biologic activity related to cell restitution in vitro; (2) if exogenous administration of recombinant human MUC17-CRD1-L-CRD2 proteins stimulated cell restitution processes in vitro; and (3) determine if recombinant human MUC17-CRD1-L-CRD2 enhances healing of experimental colitis in vivo. The findings from these studies begin to describe a CRD1-L-CRD2 unit from human MUC17 that is a candidate for further development as a potential mucosal restitution agent for the treatment of inflammatory bowel disease and other mucosal disorders.
Section snippets
Recombinant proteins
Two MUC17 recombinant proteins were synthesized with either a GST or poly-His tag, as described previously (Ho et al., 2010). The first is labeled human MUC17-CRD1-Linker (L)-CRD2-GST (R-1 through S-260) containing a N-terminal GST tag, with the sequence: (glutathione-S-transferase)-R T T T C F G D G C Q N T A S R C K N G G T W D G L K C Q C P N L Y Y G E L C E E V V S S I D I G P P E T I S A Q M E L T V T V T S V K F T E E L K N H S S Q E F Q E F K Q T F T E Q M N I V Y S G I P E Y V G V N I T
Characterization of MUC17 knock down cell line LSsi
The LS174T colon cells exhibited high levels of endogenous MUC17 mRNA and therefore was chosen to create a stably transfected cell line with MUC17 siRNA in order to demonstrate whether endogenous MUC17 mucin may play a role in cellular behavior and processes related to cell restitution. Both Western blot and RT-PCR screening for MUC17 in LSsi and LSscram showed decreased MUC17 protein and mRNA expression in LSsi as compared to LSscram (Fig. 2). LSsi and LSscram cells also exhibited
Discussion
Membrane-bound mucins such as MUC17 are highly expressed on the surface of intestinal cells, but to date little is known about their function. We demonstrate that inhibition of expression of endogenous MUC17 mucin in a high-MUC17 expressing cell line resulted in an altered morphology, reduced cellular aggregation, increased susceptibility to apoptosis, and reduced spontaneous cell migration, indicating the potential importance of endogenous membrane-bound mucins in cellular processes related to
Acknowledgements
This study was supported by a VA Merit Review grant (SBH), NIH STTR grant G1 R43 DK072629-01 (SBH, RLH), NIH center grant (DK080506) (SBH), NIH CA78590 (SKB), and the Research Service of the Department of Veterans Affairs.
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