Inactivation of G-protein-coupled receptor 48 (Gpr48/Lgr4) impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway - PubMed (original) (raw)

Inactivation of G-protein-coupled receptor 48 (Gpr48/Lgr4) impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway

Huiping Song et al. J Biol Chem. 2008.

Abstract

G-protein-coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation, and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48-/- mice and found that Gpr48-/- fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119pos population) in Gpr48-/- fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real-time PCR analyses showed that in Gpr48-/- mice both adult hemoglobin alpha and beta chains were decreased while embryonic hemoglobin chains (zeta, betaH1, and epsilony) were increased, providing another evidence for the impairment of definitive erythropoiesis. Furthermore, proliferation was suppressed in Gpr48-/- fetal liver with decreased c-Myc and cyclin D1 expression, whereas apoptosis was unaffected. ATF4, a key transcription factor in erythropoiesis, was down-regulated in Gpr48-/- fetal livers during midgestation stage through the cAMP-PKA-CREB pathway, suggesting that Gpr48 regulated definitive erythropoiesis through ATF4-mediated definitive erythropoiesis.

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Figures

FIGURE 1.

Expression of Gpr48 in fetal liver. A, RT-PCR analysis of Gpr48 mRNA levels from E13.5 Gpr48 wild-type (+/+), heterozygous (+/-), and homozygous (-/-) fetal livers, showing no Gpr48 expression in Gpr48-/- fetal liver. B and C, LacZ staining of E15.5 Gpr48 wild-type (+/+), heterozygous (+/-), and homozygous (-/-) fetal liver sections.Gpr48+/- and _Gpr48_-/- fetal liver showed positive staining in liver (upper right panel, B, magnification 100×) and at cell surface membrane (lower right panel, C, magnification 1000×). Sections were counterstained with eosin.

FIGURE 2.

Defects of Gpr48-**/**- mice in body size and fetal liver. _A, Gpr48_-/- fetal mice at E14.5 showed significantly decreased liver weight and the ratio of liver weight/body weight. _a, Gpr48_-/- fetal liver weight was decreased ∼1.68-fold compared with their wild-type littermates (p < 0.001). b, the ratio of liver weight/body weight was decreased ∼1.25-fold in _Gpr48_-/- mice compared with wild-type littermates (p < 0.02). _B, Gpr48_-/- mice embryos and livers (right) during E12.5-E15.5 are paler and smaller than wild-type. E16.5 _Gpr48_-/- embryos show smaller in size but similar in color compared with wild-type fetuses.

FIGURE 3.

**Significant decrease of erythroid precursor cells and erythroblast islands in Gpr48-/**- fetal liver by histological analysis. _Gpr48_-/- fetal liver showed significant decrease of definitive erythroid precursor cells and erythroblast islands from E12.5 to E15.5 (A-D). Erythropoietic precursor cells are indicated by yellow arrows, hepatocytes by green arrows, and erythroblast islands by orange arrows (magnification, 1000×).

FIGURE 4.

**Increased nucleated erythrocytes at midgestation of_Gpr48_-/**- mice. A, blood smears from E13.5-E16.5 embryos were stained with Wright-Giemsa stain. _Gpr48_-/- embryos (right) of E13.5-E15.5 showed increased percentage of nucleated erythrocytes compared with the samples from their wild-type littermates (left), but no significant difference from E16.5 samples between _Gpr48_-/- and their wild-type littermates (magnification, 1000×). B, relative number in percentage of nucleated erythrocytes from blood smear slides. E13.5 and E14.5 Gpr48_-/- embryos showed ∼2.5-fold and 1.8-fold increase in nucleated erythrocytes percentage compared with wild-type, respectively (p < 0.03). E12.5 and E16.5 littermates showed little differences in percentage of nucleated erythrocytes between wild-type and null embryos (_p_ > 0.05). C and_D, real-time PCR of embryonic hemoglobin chains (ζ, βh1, and εy) in E13.5 blood cell samples (C) and adult hemoglobin chains (α and β) in E13.5 livers (D). E, the cell size of erythrocytes was reduced by Gpr48 inactivation in E13.5 fetal livers. Ter119pos cells in _Gpr48_-/- mice bone marrow displayed a reduction in cell size compared with those in their wt littermates (_x_-axis represents the cell size). Green in the histogram represents Ter119-positive cells (mature red blood cells). Similar reduction of red blood cell size was observed from adult_Gpr48_-/- cells. However, the cell number did not show significant decrease in _Gpr48_-/- null mice.

FIGURE 5.

Flow cytometry analysis of fetal liver hematopoietic cells from E13. 5_Gpr48_+/+ **and_Gpr48_-/**- embryos. Cell suspensions from E13.5 fetal livers were labeled with anti-c-kit, anti-CD34, anti-CD44, or anti-Ter119-specific antibody. The populations of Ter119-positive cells, which represent definitive erythrocytes in _Gpr48_-/- embryos, were markedly decreased.

FIGURE 6.

**Reduced cell proliferation in_Gpr48_-/**- fetal liver. A, E13.5 Gpr48_-/- fetal liver showed dramatically decreased number of positive cells (dark brown) compared with wild-type sections using PCNA by immunohistochemistry. Hematoxylin stain was performed as counterstain (magnification, 1000×). B, proliferation assays with BrdUrd labeling demonstrated that_Gpr48_-/- E13.5 fatal liver have marked decrease in the number of proliferating cells (BrdUrd-positive sells, brown color). Sections were counterstained by methylene green (magnification, 1000×).C, TUNEL assays of E13.5 fetal liver showed no significant differences in the number of positive cells (brown) between_Gpr48_-/- (left panel) and_Gpr48+/+ fetal livers. Sections were counterstained by methylene green (magnification, 1000×).

FIGURE 7.

**Down-regulation of c-Myc and Cyclin D1 in_Gpr48_-/**- fetal liver. A, Western blot analysis showed that protein levels of c-Myc and Cyclin D1 were both decreased in Gpr48_-/- fetal livers compared with that in the wild type. Actin was used as an internal control. B, fetal liver sections from E13.5_Gpr48+/+ and _Gpr48_-/- embryos were stained with anti-c-Myc antibody. Significant decrease of c-Myc protein level was observed in _Gpr48_-/- fetal liver (brown color, magnification, 1000×). C, decreased protein levels of Cyclin D1 in _Gpr48_-/- fetal liver compared with wild-type. Fetal liver sections from E13.5 Gpr48+/+ and_Gpr48_-/- embryos were stained with anti-Cyclin D1 antibody (brown color, magnification, 1000×).

FIGURE 8.

**Down-regulation of ATF4 in_Gpr48_-/**- fetal liver. A, RT-PCR showed that ATF4 mRNA was dramatically decreased in _Gpr48_-/- livers during E13.5-E15.5, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. B, real-time PCR showed significantly reduced mRNA levels of ATF4 in _Gpr48_-/- livers during E13.5-E15.5, and β-actin used as an internal control. C, the protein expression level of ATF4 was significantly decreased at E13.5, E14.5, and E15.5 in_Gpr48_-/- livers by Western blot. Actin was used as an internal control. D, the expression level of ATF4 was markedly reduced in E13.5 _Gpr48_-/- fetal livers by immunocytochemistry staining. Sections were stained with anti-ATF4-specific antibody. The number of positive cells (brown) was markedly reduced compared with wild type. Hematoxylin was used as counterstain (1000×).E, Gpr48 regulates ATF4 promoter through the cAMP-PKA-CREB pathway. PKA inhibitor PKI strongly inactivated the ATF4 1.2k promoter activity induced by a constitutively activated mutant Gpr48 (T755I). The activation of ATF4 by the active Gpr48 mutant was also markedly inhibited by PKA-specific inhibitor H89, suggesting Gpr48 activates ATF4 through activation of the PKA pathway.

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Inactivation of G-protein-coupled receptor 48 (Gpr48/Lgr4) impairs definitive erythropoiesis at midgestation through down-regulation of the ATF4 signaling pathway - PubMed (original) (raw)