Carbachol activates protein kinase C in dispersed gastric chief cells.
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ABSTRACT:
SUBMITTER: Raffaniello RD
PROVIDER: S-EPMC1138116 | biostudies-other | 1994 May
REPOSITORIES: biostudies-other
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Project description:Gastrointestinal tract organs harbor reserve cells, which are endowed with cellular plasticity and regenerate functional units in response to tissue damage. However, whether the reserve cells in gastrointestinal tract exist as long-term quiescent cells remain incompletely understood. In the present study, we systematically examine H2b-GFP label-retaining cells and identify a long-term slow-cycling population in the gastric corpus but not in other gastrointestinal organs. The label-retaining cells, which reside near the basal layers of the corpus, comprise a subpopulation of chief cells. The identified quiescent cells exhibit induction of Atf4 and its target genes including Atf3, a marker of paligenosis, and activation of the unfolded protein response, but do not show elevated expression of Troy, Lgr5, or Mist. External damage to the gastric mucosa induced by indomethacin treatment triggers proliferation of the quiescent Atf4+ population, indicating that the gastric corpus harbors a specific cell population that is primed to facilitate stomach regeneration.
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Project description:Hierarchically organized tissues, such as hematopoietic systems, muscle, or skin harbor deeply quiescent stem cells which start proliferating in response to external insults. In contrast, it remains obscure whether similar quiescent cells exist in epithelia of digestive organs. Here we identified a deeply quiescent population in gastric corpus but not in other gastrointestinal organs after systematic examination of H2b-GFP label-retaining cells. The label-retaining cells in corpus epithelia belonged to a subpopulation of chief cells that were located near basal layers of corpus and did not overlap with Troyhigh, Lgr5high, or Misthigh cell population. The identified quiescent cells were marked with activation of Atf4 and unfolded protein response. External damages by indomethacin treatment triggered proliferation of the quiescent populations, indicating that chief cells of gastric corpus harbor deeply quiescent reserve cells with high levels of internal stress response activity.
2023-10-31 | GSE213755 | GEO
Project description:AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.
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Project description:ATF4 marks quiescent reserve cells of gastric chief cells
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Project description:Activation of the p21-activated kinase 1 (PAK1) is achieved through a conformational change that converts an inactive PAK1 dimer to an active monomer. In this paper, we show that this change is necessary but not sufficient to activate PAK1 and that it is, rather, required for CK2-dependent PAK1(S223) phosphorylation that converts a monomeric PAK1 into a catalytically active form. This phosphorylation appears to be essential for autophosphorylation at specific residues and overall activity of PAK1. A phosphomimetic mutation (S223E) bypasses the requirement for GTPases in PAK1 activation, whereas the constitutive activity of the PAK1 mutant (PAK1(H83,86L)), postulated to mimic GTPase-induced structural changes, is abolished by inhibition of S223 phosphorylation. Thus, S223 is likely accessible to CK2 upon conformational changes of PAK1 induced by GTPase-dependent and GTPase-independent stimuli, suggesting that S223 phosphorylation may play a key role in the final step of the PAK1 activation process. The physiological significance of this phosphorylation is reinforced by the observations that CK2 is responsible for epidermal growth factor-induced PAK1 activation and that inhibition of S223 phosphorylation abrogates PAK1-mediated malignant transformation of prostate epithelial cells. Taken together, these findings identify CK2 as an upstream activating kinase of PAK1, providing a novel mechanism for PAK1 activation.
| S-EPMC3771959 | biostudies-literature
Project description:Background & aimsAdult zymogen-producing (zymogenic) chief cells (ZCs) in the mammalian gastric gland base are believed to arise from descending mucous neck cells, which arise from stem cells. Gastric injury, such as from Helicobacter pylori infection in patients with chronic atrophic gastritis, can cause metaplasia, characterized by gastric cell expression of markers of wound-healing; these cells are called spasmolytic polypeptide-expressing metaplasia (SPEM) cells. We investigated differentiation and proliferation patterns of neck cells, ZCs, and SPEM cells in mice.MethodsC57BL/6 mice were given intraperitoneal injections of high-dose tamoxifen to induce SPEM or gavaged with H pylori (PMSS1) to induce chronic gastric injury. Mice were then given pulses of 5-bromo-2'-deoxyuridine (BrdU) in their drinking water, followed by chase periods without BrdU, or combined with intraperitoneal injections of 5-ethynyl-2'-deoxyuridine. We collected gastric tissues and performed immunofluorescence and immunohistochemical analyses to study gastric cell proliferation, differentiation, and turnover.ResultsAfter 8 weeks of continuous BrdU administration, fewer than 10% of homeostatic ZCs incorporated BrdU, whereas 88% of neck cells were labeled. In pulse-chase experiments, various chase periods decreased neck cell label but did not increase labeling of ZCs. When mice were given BrdU at the same time as tamoxifen, more than 90% of cells were labeled in all gastric lineages. After 3 months' recovery (no tamoxifen), ZCs became the predominant BrdU-labeled population, whereas other cells, including neck cells, were mostly negative. When we tracked the labeled cells in such mice over time, we observed that the proportion of BrdU-positive ZCs remained greater than 60% up to 11 months. In mice whose ZCs were the principal BrdU-positive population, acute injury by tamoxifen or chronic injury by H pylori infection resulted in SPEM cells becoming the principal BrdU-positive population. After withdrawal of tamoxifen, BrdU-positive ZCs reappeared.ConclusionsWe studied mice in homeostasis or with tamoxifen- or H pylori-induced SPEM. Our findings indicated that mucous neck cells do not contribute substantially to generation of ZCs during homeostasis and that ZCs maintain their own census, likely through infrequent self-replication. After metaplasia-inducing injury, ZCs can become SPEM cells, and then redifferentiate into ZCs on injury resolution.
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Project description:The orderly differentiation of cell lineages within gastric glands is regulated by a complicated interplay of local mucosal growth factors and hormones. Histamine secreted from enterochromaffin-like cells plays an important role in not only stimulated gastric acid secretion but also coordination of intramucosal growth and lineage differentiation. We have examined histidine-decarboxylase (HDC)-deficient mice, which lack endogenous histamine synthesis, to evaluate the influence of histamine on differentiation of fundic mucosal lineages and the development of metaplasia following induction of acute oxyntic atrophy. Stomachs from HDC-deficient mice and wild-type mice were evaluated at 8 wk and 12 mo of age. DMP-777 was administrated orally to 6-wk-old mice for 1 to 14 days. Sections of gastric mucosa were stained with antibodies against Mist1, intrinsic factor, H/K-ATPase, trefoil factor 2 (TFF2), chromogranin A, and Ext1 and for the cell cycle marker phospho-histone H3. HDC-deficient mice at 8 wk of age demonstrated a prominent increase in chief cells expressing Mist1 and intrinsic factor. Importantly Mist1-positive mature chief cells were present in the midgland region as well as at the bases of fundic glands, indicating a premature differentiation of chief cells. Mice dually deficient for both HDC and gastrin showed a normal distribution of chief cells in fundic glands. Treatment of HDC-deficient mice with DMP-777 led to loss of parietal cells and an accelerated and exaggerated emergence of mucous cell metaplasia with the presence of dual intrinsic factor and TFF2-expressing cells throughout the gland length, indicative of the emergence of spasmolytic polypeptide-expressing metaplasia (SPEM) from chief cells. These findings indicate that histamine, in concert with gastrin, regulates the appropriate differentiation of chief cells from mucous neck cells as they migrate toward the bases of fundic glands. Nevertheless, histamine is not required for emergence of SPEM following acute oxyntic atrophy.
| S-EPMC2697940 | biostudies-literature
Project description:Engineered overexpression of protein kinase Calpha (PKCalpha) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCalpha (Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370) revealed that alpha6-tubulin is phosphorylated in cells expressing traceable PKCalpha and in vitro by wild type PKCalpha. Gain-of-function, single site mutations (Ser-->Asp) were constructed at each PKC consensus site in alpha6-tubulin (Ser158, Ser165, Ser241, and Thr337) to simulate phosphorylation. Following expression of each construct in MCF-10A cells, motility assays identified Ser165 as the only site in alpha6-tubulin whose pseudophosphorylation reproduced the motile behavior engendered by PKCalpha. Expression of a phosphorylation-resistant mutant (S165N-alpha6-tubulin) resulted in suppression of MCF-10A cell motility stimulated either by expression of PKCalpha or by treatment with PKCalpha-selective activator diacylglycerol-lactone. MCF-10A cells treated with diacylglycerol-lactone showed strong phosphorylation of endogenous alpha-tubulin that could be blocked when S165N-alpha6-tubulin was expressed. The S165N mutant also inhibited intrinsically motile human breast tumor cells that express high endogenous PKCalpha levels (MDA-MB-231 cells) or lack PKCalpha and other conventional isoforms (MDA-MB-468 cells). Comparison of Myc-tagged wild type alpha6-tubulin and S165N-alpha6-tubulin expressed in MDA-MB-468 cells demonstrated that Ser165 is also a major site of phosphorylation for endogenously active, nonconventional PKC isoforms. PKC-stimulated motility of MCF-10A cells was nocodazole-sensitive, thereby implicating microtubule elongation in the mechanism. These findings support a model in which PKC phosphorylates alpha-tubulin at Ser165, leading to microtubule elongation and motility.
| S-EPMC2719404 | biostudies-literature
Project description:Injury and inflammation in the gastric epithelium can cause disruption of the pathways that guide the differentiation of cell lineages, which in turn can cause persistent alterations in differentiation patterns, known as metaplasia. Metaplasia that occurs in the stomach is associated with increased risk for cancer. Methods for isolating distinct gastric epithelial cell populations would facilitate dissection of the molecular and cellular pathways that guide normal and metaplastic differentiation. Here, we identify alanyl aminopeptidase (CD13) as a specific surface marker of zymogenic chief cells (ZCs) in the gastric epithelium. We show that 1) among gastric epithelial cells alanyl aminopeptidase expression is confined to mature ZCs, and 2) its expression is lost en route to metaplasia in both mouse and human stomachs. With this new marker coupled with new techniques that we introduce for dissociating gastric epithelial cells and overcoming their constitutive autofluorescence, we are able to reliably isolate enriched populations of ZCs for both molecular analysis and for the establishment of ZC-derived ex vivo gastroid cultures.
| S-EPMC4683299 | biostudies-literature
Project description:Atypical protein kinase iota (PKCiota) is a key organizer of the apical domain in epithelial cells. Ezrin is a cytosolic protein that, upon activation by phosphorylation of T567, is localized under the apical membrane where it connects actin filaments to membrane proteins and recruits protein kinase A (PKA). To identify the kinase that phosphorylates ezrin T567 in simple epithelia, we analyzed the expression of active PKC and the appearance of T567-P during enterocyte differentiation in vivo. PKCiota phosphorylated ezrin on T567 in vitro, and in Sf9 cells that do not activate human ezrin. In CACO-2 human intestinal cells in culture, PKCiota co-immunoprecipitated with ezrin and was knocked down by shRNA expression. The resulting phenotype showed a modest decrease in total ezrin, but a steep decrease in T567 phosphorylation. The PKCiota-depleted cells showed fewer and shorter microvilli and redistribution of the PKA regulatory subunit. Expression of a dominant-negative form of PKCiota also decreased T567-P signal, and expression of a constitutively active PKCiota mutant showed depolarized distribution of T567-P. We conclude that, although other molecular mechanisms contribute to ezrin activation, apically localized phosphorylation by PKCiota is essential for the activation and normal distribution of ezrin at the early stages of intestinal epithelial cell differentiation.
| S-EPMC2293289 | biostudies-literature