The hyaluronic acid receptor CD44 coordinates normal and metaplastic gastric epithelial progenitor cell proliferation

Abstract

The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44(+) population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK → CD44 → STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.

Keywords: Cd44; ERK; Gastric Cancer; Hyaluronan; PEP-1; Signaling; Stem Cells; Tamoxifen; U0126; WP1066.

FIGURE 1.

CD44 labels undifferentiated cells in the normal stem cell zone, i.e. the isthmus, of the gastric unit, and its loss stunts basal rates of proliferation. In the normal mouse gastric unit, CD44 labeled small, distinct cells in the isthmus region (A). Mice lacking the Cd44 gene have about half the number of proliferating cells per gastric unit compared with WT controls (B and C). PEP-1 is a peptide inhibitor that blocks the interaction between hyaluronic acid and CD44. Treatment with PEP-1 for 5 weeks reduced basal rates of proliferation (D) and resulted in lower numbers of pit cells (E), similar to the Cd44^−/− animals (E). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

H. pylori infection causes parietal cell atrophy and expansion of CD44 expression. Hematoxylin- and eosin-stained sections of wild-type, uninfected mice show healthy parietal cells (A, left, green arrowhead), whereas those infected for 8 weeks with the cag⁺ PMSS1 strain of H. pylori showed diffuse loss of parietal cells (A, right). The gastric unit is largely replaced with metaplastic cells (A, right; brown arrowheads), and only a few parietal cells remained (A, right; green arrowheads). CD44 also labeled occasional immune cells infiltrating interglandular regions (B, right; beige arrowheads). CD44 is expressed in the uninfected gastric epithelium in the isthmus (B, blue box, and C, orange arrowheads) but expands to the base of the unit upon infection with H. pylori (B, right, yellow box). In uninfected mice, the zymogenic cells did not express CD44 (C, left, white arrowheads); however, upon infection, they became metaplastic and expressed neck cell markers such as GSII (red) as well as CD44 (C, right, yellow arrowheads). In mice infected with H. pylori for a shorter time period of 4 weeks (D), CD44 expansion can be seen to extend from the isthmus (purple box) and into the base (orange box) in some gastric units. Exemplar CD44⁺ cells are marked with purple arrowheads (D, insets); gastric units are outlined by dashed white line.

FIGURE 3.

CD44 expands and labels proliferating cells upon parietal cell atrophy and is required for this injury induced expansion of progenitor cells. Hematoxylin- and eosin-stained sections of wild-type mice at 3 days (A, left) after intraperitoneal injection of vehicle and at 12 h (A, middle) and 3 days (A, right) after intraperitoneal injection of 5 mg/20-g body weight of tamoxifen. Wild-type mouse stomach treated with vehicle showed normal stomach epithelium, whereas those injected with tamoxifen showed a progressive loss of PCs. PC loss is coupled with an expansion in proliferation, measured by BrdU incorporation (stained in red) at 12 h (B, middle; C) and 3 days (B, right, C) after tamoxifen treatment compared with vehicle controls (B, left, C). Cyclin D1 and PCNA, which are markers of proliferation, were also increased on PC atrophy at 12 h and day 3 by Western blot of whole corpus stomach regions (D). The blot also shows that CD44 expression increases upon atrophy. A CD44⁺ epithelial population started expanding from the time point (6 h) when PCs first began to die (E) and reached the base of the unit by day 3 (E). The number of CD44⁺ epithelial cells expanded ∼3–5-fold during this time, as shown by multiple FACS experiments (F). One of the FACS plots graphed in F is shown in G. Many CD44-expressing cells co-stain with Ki67 after treatment with tamoxifen (H, the yellow box is magnified in the inset at the right).

FIGURE 4.

CD44 is necessary for elevating the rate of progenitor cell proliferation upon induction of atrophy. PEP-1 blockade of CD44 activation halved atrophy-induced proliferation (B and C), measured by BrdU incorporation (red, B), whereas parietal cells died upon PEP-1 inhibition of CD44 (A).

FIGURE 5.

CD44 regulates gastric progenitor cell proliferation through STAT3. STAT3 was activated by tyrosine phosphorylation after atrophy induction with tamoxifen (A). Immunoprecipitation (IP) with antibody against STAT3 followed by Western blot for CD44 showed increased association between CD44 and STAT3 during tamoxifen-induced atrophy compared with controls; also, phosphorylated STAT3 was pulled down only during tamoxifen-induced atrophy (B). Inhibition of STAT3 and CD44 functions (using WP1066 and PEP-1, respectively) caused decreased cyclin D1 expression (C and E) and proliferation as quantified in tissue using BrdU (D); D1, 1 day post tamoxifen; D2, 2 days. CD44 levels were not affected by STAT3 inhibition, whereas pSTAT3 was significantly reduced upon CD44 inhibition with PEP-1 (C and E). CD44 expression was reduced upon blocking interaction with its HA ligand by PEP-1 (C).

FIGURE 6.

ERK signaling is activated early upon induction of injury and is required to induce CD44. Shown are Western blots of candidate signaling pathways that might be involved in increasing the proliferative response after PC atrophy by tamoxifen (A). PLC, phospholipase C. Of the pathways analyzed, only ERK signaling showed a dramatic increase in activation after tamoxifen compared with vehicle controls. ERK1/2 were tyrosine-phosphorylated soon after PC damage ensued (6 h after tamoxifen treatment); EGR1 and CD44, downstream targets of ERK signaling, were already increased in expression at this early time point (B). Co-injection of tamoxifen with U-0126, a specific inhibitor of ERK phosphorylation, muted the metaplastic induction of pERK along with downstream targets, EGR1 and CD44, by Western blot (C) and muted the proliferative response shown by BrdU immunostaining in red (D), quantified in E.

FIGURE 7.

pERK labels metaplasia-associated cells in mice and humans. pERK labeled nuclei of isthmal progenitor cells (orange arrowheads) at 6 h of tamoxifen treatment (A and B), whereas there was no pERK signal in vehicle-treated controls. PCs did not stain positive for pERK in either vehicle- or tamoxifen-treated mice. Gastric units of human patients showed focal intestinal metaplasia (C, right) in a region with mixed gastric and intestinal differentiation (magnified in E), indicating transitional/early metaplasia (goblet cell is marked with a yellow arrowhead) stain positive for pERK (green arrowheads). Shown is quantification of pERK expression in human IM patients (n = 3) displaying such a transitional epithelium by counting the number of pERK⁺ cells in the intestinal metaplasia (IM) regions and in residual gastric-differentiation regions “Adjacent to IM” epithelium versus those in normal humans showed a dramatic increase in pERK⁺ cells in IM regions, with a significant increase in pERK⁺ cells even in gastric regions adjacent to IM when compared with normal human patients (D). IHC, immunohistochemistry.

FIGURE 8.

Model for stem/progenitor cell renewal during normal and atrophic injury conditions. The isthmal stem cell (SC) is self-renewing and also gives rise to acid-secreting PCs among other cell-types within the stomach. Upon atrophy of PCs by toxins or H. pylori infection, the stem cells activate ERK to ramp up proliferation. ERK activation is required for expanded CD44 expression and the association between CD44 and pSTAT3, which in turn up-regulates cyclin D1 to drive proliferation.