Doctor of Philosophy, University of California Berkeley (2009)
Eugene Butcher, Postdoctoral Faculty Sponsor
Lymphatic fluid is a plasma filtrate that can be viewed as having biological activity through the passive accumulation of molecules from the interstitial fluid. The possibility that lymphatic fluid is part of an active self-contained signaling process that parallels the endocrine system, through the activation of G-protein coupled receptors (GPCR), has remained unexplored. We show that the GPCR lysophosphatidic acid 5 (LPA5) is found in sensory nerve fibers expressing calcitonin gene-related peptide (CGRP) that innervate the lumen of lymphatic lacteals and enteric nerves. Using LPA5 as a model for nutrient-responsive GPCRs present on sensory nerves, we demonstrate that dietary protein hydrolysate (peptone) can induce c-Fos expression in enterocytes and nerves that express LPA5. Mesenteric lymphatic fluid (MLF) mobilizes intracellular calcium in cell models expressing LPA5 upon feeding in a time- and dose-dependent manner. Primary cultured neurons of the dorsal root ganglia expressing CGRP are activated by MLF, which is enhanced upon LPA5 overexpression. Activation is independent of the known LPA5 agonists, lysophosphatidic acid and farnesyl pyrophosphate. These data bring forth a pathway for the direct stimulation of sensory nerves by luminal contents and interstitial fluid. Thus, by activating LPA5 on sensory nerves, MLF provides a means for known and yet to be identified constituents of the interstitial fluid to act as signals to comprise a "neurolymphocrine" system.
View details for DOI 10.1152/ajpgi.00433.2013
View details for PubMedID 24578341
Dendritic cells (DCs) that orchestrate mucosal immunity have been studied in mice. Here we characterized human gut DC populations and defined their relationship to previously studied human and mouse DCs. CD103(+)Sirpα(-) DCs were related to human blood CD141(+) DCs and to mouse intestinal CD103(+)CD11b(-) DCs and expressed markers of cross-presenting DCs. CD103(+)Sirpα(+) DCs aligned with human blood CD1c(+) DCs and mouse intestinal CD103(+)CD11b(+) DCs and supported the induction of regulatory T cells. Both CD103(+) DC subsets induced the TH17 subset of helper T cells, while CD103(-)Sirpα(+) DCs induced the TH1 subset of helper T cells. Comparative analysis of transcriptomes revealed conserved transcriptional programs among CD103(+) DC subsets and identified a selective role for the transcriptional repressors Bcl-6 and Blimp-1 in the specification of CD103(+)CD11b(-) DCs and intestinal CD103(+)CD11b(+) DCs, respectively. Our results highlight evolutionarily conserved and divergent programming of intestinal DCs.
View details for DOI 10.1038/ni.2768
View details for Web of Science ID 000328800500017
View details for PubMedID 24292363
P2Y5 is a G protein-coupled receptor that binds and is activated by lysophosphatidic acid (LPA). We determined that P2Y5 transcript is expressed along the intestinal mucosa and investigated the intracellular pathways induced by P2Y5 activation, which could contribute to LPA effects on intestinal cell adhesion. P2Y5 heterologously expressed in CHO and small intestinal hBRIE 380i cells was activated by LPA resulting in an increase in intracellular calcium ([Ca(2+)](i)) when the cells concurrently expressed G(alpha)(Delta6qi5myr). P2Y5 activation also increased the phosphorylation of ERK1/2 that was sensitive to pertussis toxin. Together these indicate that P2Y5 activation by LPA induces an increase in [Ca(2+)](i) and ERK1/2 phosphorylation through G(alpha)(i). We discovered that P2Y5 was activated by farnesyl pyrophosphate (FPP) without a detectable change in [Ca(2+)](i). The activation of P2Y5 by LPA or FPP induced the activity of a serum response element (SRE)-linked luciferase reporter that was inhibited by the RGS domain of p115RhoGEF, C3 exotoxin, and Y-27632, suggesting the involvement of G(alpha)(12/13), Rho GTPase, and ROCK, respectively. However, only LPA-mediated induction of SRE reporter activity was sensitive to inhibitors targeting p38 MAPK, PI3K, PLC, and PKC. In addition, only LPA transactivated the epidermal growth factor receptor, leading to an induction of ERK1/2 phosphorylation. These observations correlate with our subsequent finding that P2Y5 activation by LPA, and not FPP, reduced intestinal cell adhesion. This study elucidates a mechanism whereby LPA can act as a luminal and/or serosal cue to alter mucosal integrity.
View details for DOI 10.1152/ajpgi.00191.2009
View details for Web of Science ID 000270141400003
View details for PubMedID 19679818
In the intestinal lumen, protein hydrolysate increases the transcription and release of cholecystokinin (CCK) from enteroendocrine cells of the duodenal-jejunal mucosa. Our recent discovery that a G protein-coupled receptor, GPR93, is activated by dietary protein hydrolysate causing induced intracellular calcium-mediated signaling events in intestinal epithelial cells raises a possibility that GPR93 might be involved in the protein hydrolysate induction of CCK expression and/or secretion. Using the enteroendocrine STC-1 cells as a model, the present study demonstrates that increasing expression of GPR93 amplifies the peptone induction of endogenous CCK mRNA levels. A similar increase in CCK transcription, indicated by the luciferase reporter activity driven by an 820-bp CCK promoter, is also observed in response to peptone at a dose as little as 6.25 mg/ml, but not to lysophosphatidic acid (LPA), an agonist of GPR93. We discovered that the upregulation of CCK transcription involves ERK1/2, PKA, and calmodulin-dependent protein kinase-mediated pathways. Additionally, GPR93 activation by peptone induces a response in CCK release at 15 min, which continues over a 2-h period. The cAMP level in STC-1 cells overexpressing GPR93 is induced at a greater extent by peptone than by LPA, suggesting a possible explanation of the different effects of peptone and LPA on CCK transcription and secretion. Our data indicate that GPR93 can contribute to the observed induction of CCK expression and secretion by peptone and provide evidence that G protein-coupled receptors can transduce dietary luminal signals.
View details for DOI 10.1152/ajpgi.00516.2006
View details for Web of Science ID 000247935800020
View details for PubMedID 17290006
G protein-coupled receptors (GPCRs) have the potential to play a role as molecular sensors responsive to luminal dietary contents. Although such a role for GPCRs has been implicated in the intestinal response to protein hydrolysate, no GPCR directly involved in this process has been previously identified. In the present study, for the first time, we identified GPR93 expression in enterocytes and demonstrated its activation in these cells by protein hydrolysate with EC50 of 10.6 mg/ml as determined by the induction of intracellular free Ca2+. In enterocytes, GPR93 was synergistically activated by protein hydrolysate in combination with an agonist, oleoyl-l-alpha-lysophosphatidic acid (LPA), which activated the receptor in these enterocytes with EC50 of 7.9 nM. The increased intracellular Ca2+ by GPR93 activation was observed without the addition of a promiscuous Galpha protein and was pertussis toxin sensitive, which suggests Galpha(q)- and Galpha(i)-mediated pathways. Activated GPR93 also induced pertussis toxin-sensitive ERK1/2 phosphorylation. Both nuclear factor of activated T cells and 12-O-tetradecanoylphorbol 13-acetate responsive elements reporter activities were induced by protein hydrolysate in cells exogenously expressing GPR93. The peptidomimetic cefaclor by itself did not activate GPR93 but potentiated the protein hydrolysate response and further amplified the synergistic enhancement of GPR93 activation by protein hydrolysate and LPA. These data suggest that, physiologically, the composition of stimuli might determine GPR93 activity or its sensitivity toward a given activator and suggest a new mechanism of the regulation of mucosal cell proliferation and differentiation and hormonal secretion by dietary products in the lumen.
View details for DOI 10.1152/ajpgi.00295.2006
View details for Web of Science ID 000243399800012
View details for PubMedID 16935853
Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.
View details for DOI 10.1074/jbc.M408858200
View details for Web of Science ID 000226025100017
View details for PubMedID 15504731