Epithelial cells assemble into mucosal sheets and act as the barrier between the external and internal milieu. To maintain this functional barrier, intestinal epithelial cells must establish segregated apical and basolateral domains. Alterations in these processes through losses in polarity can lead to neoplasia, while loss of proper microvillar assembly or maintenance can lead to malabsorbtion of nutrients and diarrheal disease. Our recent studies have demonstrated that vesicle trafficking proteins critically regulate the processes required for establishment and maintenance of apical polarity. Rab11-Family Interacting Proteins (Rab11-FIPs) mediate both directional trafficking and the establishment of apical polarity. The establishment of apical polarity involves proper phosphorylation of Rab11-FIP2 by the polarity-associated kinase MARK2/Par1b. We have recently demonstrated that Rab11-FIP1B/C is also a substrate for MARK2. While previous investigations have noted the importance of MARK2 phosphorylation in epithelial polarity, no studies have examined how MARK2- dependent phosphorylation events could influence intestinal epithelial cell polarity. In intestinal cells, we also have demonstrated that loss of Rab25, an epithelial-specific small GTP binding protein, in Rab25 KO mice promotes intestinal and colonic neoplasia and knockdown of Rab25 expression in CaCo-2 cells promotes a loss in polarized function and assumption of a more invasive phenotype. Rab25 expression is decreased in human colon cancers regardless of stage, suggesting a role for Rab25 loss in the early stages of carcinogenesis. In both the Rab25 KO mice and CaCo-2 cells, loss of Rab25 is associated with mis-trafficking of ?1-integrin. In CaCo-2 cells, knockdown of Rab25 expression induces deficits in polarized function with both mistrafficking of integrins as well as decreases in ?5-integrin transcription. We have hypothesized that components of plasma membrane recycling systems, Rab25 and Rab11-FIP1B/C and Rab11-FIP2, are critical mediators of both the establishment and maintenance of polarity and that defects in these pathways predispose to early carcinogenesis. To investigate our hypothesis we will pursue three specific aims: First, we will determine how Rab25 regulates intestinal polarity through regulation of ETV4-dependent gene transcription. Second, we will identify the roles of Rab11-FIP proteins in regulating polarity in intestinal cells. These studies will utilize novel cell culture models and phosphorylation site-specific antibodies. Third, we will determine the effects of the loss of Rab11-FIP2 and Rab11-FIP1B/C on intestinal cell polarity and differentiation in mice and intestinal enteroids in culture. These studies will utilize novel mouse models for floxed alleles for both Rab11-FIP1B/C and Rab11-FIP2. These investigations will establish how disruption of vesicle trafficking processes that regulate the establishment and maintenance of intestinal epithelial cell polarity may lead to carcinogenesis. !

Public Health Relevance

The establishment and maintenance of polarity are essential for the proper physiology of the intestinal epithelia, and loss of polarity can lead to carcinogenesis or diarrheal disease. This proposal seeks to define the intracellular membrane trafficking pathways that are responsible for polarized intestinal epithelial function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048370-20
Application #
8719085
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Carrington, Jill L
Project Start
1994-08-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
20
Fiscal Year
2014
Total Cost
$341,294
Indirect Cost
$123,794
Name
Vanderbilt University Medical Center
Department
Surgery
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Schafer, Jenny C; Baetz, Nicholas W; Lapierre, Lynne A et al. (2014) Rab11-FIP2 interaction with MYO5B regulates movement of Rab11a-containing recycling vesicles. Traffic 15:292-308
Yu, Shiyan; Nie, Yingchao; Knowles, Byron et al. (2014) TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis. EMBO J 33:1882-95
Qi, Mingli; Williams, Janice A; Chu, Hin et al. (2013) Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex. PLoS Pathog 9:e1003278
Krishnan, Moorthy; Lapierre, Lynne A; Knowles, Byron C et al. (2013) Rab25 regulates integrin expression in polarized colonic epithelial cells. Mol Biol Cell 24:818-31
Khandelwal, Puneet; Prakasam, H Sandeep; Clayton, Dennis R et al. (2013) A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells. Mol Biol Cell 24:1007-19
Baetz, Nicholas W; Goldenring, James R (2013) Rab11-family interacting proteins define spatially and temporally distinct regions within the dynamic Rab11a-dependent recycling system. Mol Biol Cell 24:643-58
Goldenring, James R (2013) A central role for vesicle trafficking in epithelial neoplasia: intracellular highways to carcinogenesis. Nat Rev Cancer 13:813-20
Mattaloni, Stella M; Kolobova, Elena; Favre, Cristian et al. (2012) AKAP350 Is involved in the development of apical "canalicular" structures in hepatic cells HepG2. J Cell Physiol 227:160-71
Lapierre, Lynne A; Ducharme, Nicole A; Drake, Kimberly R et al. (2012) Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells. Exp Cell Res 318:103-13
Lapierre, Lynne A; Avant, Kenya M; Caldwell, Cathy M et al. (2012) Phosphorylation of Rab11-FIP2 regulates polarity in MDCK cells. Mol Biol Cell 23:2302-18

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