Small Proline Rich Protein-2 Expression and Regulation in the Caco-2 model of Intestinal Epithelial Differentiation along the Crypt-Villus Axis

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2.

The Influence of 1,25-Dihydroxyvitamin D3 on the Cross-Priming of Lymphocytic Choriomeningitis Virus Nucleoprotein

Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly alters the immunodominance hierarchy of a subsequent LCMV infection. Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter cross-priming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects.

Regulation of the Gene Encoding Thrombin-Activable Fibrinolysis Inhibitor in Non-Hepatic Cells

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like pro-enzyme that, once activated, attenuates fibrinolysis. TAFIa also possesses anti-inflammatory properties. Although liver is the main source of plasma TAFI, platelet-derived TAFI has also been reported. An alternatively spliced TAFI variant resulted from the skipping of exon 6 and a 52-base deletion in exon 10 of CPB2 mRNA (∆6+10) was described to be brain specific. This TAFI variant is reputed to possess a secretase-like activity that cleaves β-amyloid precursor protein to form β-amyloid, a process involved in the onset of Alzheimer's disease. In this thesis, we report the identification of CPB2 mRNA and TAFI protein in various vascular and inflammatory cells. Specifically, we describe the expression of CPB2 mRNA in the megakaryocytic cell lines MEG-01 and Dami, the monocytic cell line THP-1, and peripheral blood mononuclear cells. TAFI protein was detected in differentiated Dami and THP-1 cells. We next describe the effect of external stimuli such as phorbol myristate acetate (PMA) on CPB2 expression in Dami and THP-1 cells. We found that PMA treatment increases both CPB2 mRNA abundance and promoter activity in Dami cells, and decreases both CPB2 mRNA abundance and promoter activity in THP-1 cells. Deletion analysis of the CPB2 promoter indicated cell-type specific regulation of CPB2 gene expression. Finally, we evaluated the expression of alternatively spliced CPB2 mRNA variants in hepatic and non hepatic cells. We found that exon 6 skipping variants are expressed in all cell types of interest. The variant previously reported to be brain specific was also found to be expressed in platelets. We found that the alternatively spliced TAFI variants accumulated inside the cells in a non-secretable, hypoglycosylated form and showed no carboxypeptidase activity. Taken together, this thesis provides further evidence supporting the hypothesis that platelet-derived TAFI is originated from CPB2 gene expression in megakaryocytes. Moreover, our data imply a potential for site-specific anti-inflammatory control provided by macrophage-derived TAFI. Alternative splicing of the CPB2 mRNA may give rise to variants with an intracellular role, perhaps as a peptidase chaperone, and may modulate the synthesis of secretable TAFI.