MAPK signaling pathway regulates p27 phosphorylation at threonin 187 as part of the mechanism triggered by early-weaning to induce cell proliferation in rat gastric mucosa

Abstract

During rat postnatal development, gastric cell proliferation and differentiation depend on many elements, which include dietary pattern, hormones, growth factors and their signaling pathways. Among them, EGFR activity is increased through MAPK and Src cascades in response to early weaning that represents the abrupt transition from milk to solid food. We herein investigated the direct involvement of ERK pathway in the control of cell cycle progression during early weaning, and studied the specific role of p27. At 15 days, Wistar rats were separated from dams, fed with powdered chow and daily injected with PD98059 (MEK inhibitor, 300 µg/kg) or 0.5% DMSO (control). By using HE staining and immunohistochemistry for PCNA, we respectively detected mitotic (MI) and proliferative (PI) indices in 18-day-old pups, and observed that both were reduced by PD98059. As cell cycle-related proteins (cyclin E, CDK2, cyclin D1, CDK4, p21 and p27) are involved in proliferative regulation, we compared samples obtained at 17 days in the morning (17 d) and evening (17.5 d). We found that they were not altered after ERK inhibition, but cyclin D1, p21 and p27 levels changed throughout the day in the control group. As p27 activity depends on its integrity, we studied p27 phosphorylation (threonin 187), and observed that ERK inhibition reduced this process. We suggest that MAPK pathway interferes in the regulation of p27 function in the gastric mucosa during early weaning, possibly by controlling its degradation, and altogether this mechanism might contribute to the increase of epithelial proliferation at this condition.

Figure 1. PD98059 inhibits ERK1/2 phosphorylation in 17-day-old rats submitted to early weaning.

Animals were treated with 0.5% DMSO (control) or PD98059 at 300 µg/kg. Immunoblots and the respective densitometries are shown for p-ERK1/2, ERK1, p-Src and Src. Thirty µg of total protein were applied to each lane and each band is representative of one animal. β-actin was used as loading control. Relative densitometries as ratio of p-ERK/ERK1 and p-Src/Src are shown as fold change of control group and are presented by bars as means±SD (n = 3/group). *p<0.05 when comparing PD98059-treated group to control. Assays were conducted in duplicates.

Figure 2. Inhibition of MAPK phosphorylation impairs the proliferation in the gastric epithelium in early-weaned rats.

(A and B) Hematoxylin & Eosin stained sections of the gastric mucosa of 18-day-old early-weaned rats. Arrows indicate mitotic cells. Original magnification: 40×. (D and E) Immunohistochemistry for PCNA developed with DAB and counterstained with Mayer's Hematoxylin. (F) Negative control. Original magnification: 20×. (C) Mitotic Index (MI) and (G) Proliferative Index (PI) in 18-day-old early weaned rats treated with 0.5% DMSO (control) or PD98059 at 300 µg/kg. Indices are represented by bars as means±SD. MIs and PIs were obtained after counting 2,500 epithelial cells/animal, and they represent the number of mitotic or PCNA-labeled cells/total number of epithelial cells. (n) number of animals in each group. Results were analyzed statistically by Student t test. *p<0.05 when compared to the control group.

Figure 3. ERK inhibition and cell cycle-related proteins in the gastric mucosa of 17-day-old early-weaned rats.

Animals were treated daily with 0.5% DMSO (control) or PD98059 at 300 µg/kg and samples were collected in the morning (17 d) and evening (17.5 d). Immunoblots and the respective densitometries are shown for (A) p21, p27, cyclin E, CDK2, cyclin D1, CDK4 and (B) phospho-p27. Thirty µg of total protein were loaded into each lane and each band is representative of one animal. β-actin was used as loading control. Relative densitometry as ratio of β-actin (A) or total p27 (B) is shown as fold change of control group (17 d) and is presented by bars as means±SD (n = 3 in each control group and n = 4 in each PD group). Results were analyzed statistically by Two-Way ANOVA followed by Bonferroni test. Daily variation (17 and 17.5) was significantly different for p21, p27 and cyclin D1 (p<0.01). Interaction between age (17 and 17.5) and treatment was highly significant for phospho-p27 (p = 0.0071). *p<0.05 when compared to the respective control group. ^#p<0.05 when compared to the same treatment at 17 d. Assays were conducted in triplicates.