Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2 - PubMed (original) (raw)

. 2008 Apr 22;105(16):6081-6.

doi: 10.1073/pnas.0708257105. Epub 2008 Apr 18.

Jian Luo, Xuhong Cheng, Chang Jin, Xiangtian Zhou, Jia Qu, Lili Tu, Di Ai, Dali Li, Jun Wang, James F Martin, Brad A Amendt, Mingyao Liu

Affiliations

Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2

Jinsheng Weng et al. Proc Natl Acad Sci U S A. 2008.

Abstract

The development of the anterior segment of the mammalian eye is critical for normal ocular function, whereas abnormal development can cause glaucoma, a leading cause of blindness in the world. We report that orphan G protein-coupled receptor 48 (Gpr48/LGR4) plays an important role in the development of the anterior segment structure. Disruption of Gpr48 causes a wide spectrum of anterior segment dysgenesis (ASD), including microphthalmia, iris hypoplasia, irdiocorneal angle malformation, cornea dysgenesis, and cataract. Detailed analyses reveal that defective iris myogenesis and ocular extracellular matrix homeostasis are detected at early postnatal stages of eye development, whereas ganglion cell loss, inner nuclear layer thinness, and early onset of glaucoma were detected in 6-month-old Gpr48(-/-) mice. To determine the molecular mechanism of ASD caused by the deletion of Gpr48, we performed gene expression analyses and revealed dramatic down-regulation of Pitx2 in homozygous knockout mice. In vitro studies with the constitutively active Gpr48 mutant receptor demonstrate that Pitx2 is a direct target of the Gpr48-mediated cAMP-CREB signaling pathway in regulating anterior segment development, suggesting a role of Gpr48 as a potential therapeutic target of ASD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Microphthalmia, iris hypoplasia, and iridiocorneal abnormality in _Gpr48_−/− mice. (a) Microphthalmia in _Gpr48_−/− mice. (b) Iris hypoplasia demonstrated by Slit lamp examination of adult mice. _Gpr48_−/− mice have hypoplasia of the iris with a larger pupil (arrows) relative to wild-type mice under strong light exposure. (c) Midsagittal sections of the entire eye showing short iris and bigger pupil in _Gpr48_−/− (−/−) compared with wild-type mice (+/+). Detailed analysis of 24 _Gpr48_−/− and wild-type mice indicates the iris length is significantly shorter, whereas the pupil size is much bigger in _Gpr48_−/− compared with wild-type mice. (d) Smooth muscle hypoplasia and reduction in cell numbers in the iris of _Gpr48_−/− mice. The pupil part of iris in _Gpr48_−/− mice is much smaller than in wild type. The middle part of the iris in _Gpr48_−/− mice is much thinner than in wild type (H&E staining). The stroma portion in the _Gpr48_−/− iris can hardly be detected. SMA staining of the iris smooth muscle cell in adult mice demonstrates a decrease of SMA in both the pupil and the middle portions of iris muscle cells in _Gpr48_−/− mice (SMA staining). DAPI staining indicates that the number of cells was significantly reduced in both the stroma and the middle portion of the iris in _Gpr48_−/− mice (DAPI). (e) Iris myogenesis in postnatal day 4 _Gpr48_−/− mice. H&E staining shows that the iris is short and small in postnatal day 4 _Gpr48_−/− compared with wild-type mice. SMA immunofluorescence staining is weak in _Gpr48_−/− compared with wild-type mice. (f) The expression levels of key genes in myogenesis, including Mf5, Smy1D1, MyoD1, and Msc, were significantly decreased in P0 _Gpr48_−/− mice. (g) Iris–cornea adhesion and close of iridocorneal angle were detected in _Gpr48_−/− mice. (h) Iridocorneal structure anomalies in _Gpr48_−/− mice. The CB of _Gpr48_−/− mice contains less folding and fewer cells in the outer layer of the CB. The TM is compressed in _Gpr48_−/− compared with wild-type mice. C, cornea; I, iris; L, lens; P, pupil; R, retina. [Scale bars: 170 μm (c), 43 μm (d), and 85 μm (e–h).]

Fig. 2.

Fig. 2.

Cataract and ganglion cell loss in _Gpr48_−/− mice. (a) Lens opacity in _Gpr48_−/− mice. Total and partial lens opacity and iris cornea adhesion are detected in _Gpr48_−/− mice. (b) Histological analysis shows abnormal lens fibers in the lens cortical zone of _Gpr48_−/− mice. Abnormal protein deposit is also detected (arrow) (32). (c) Statistical analysis shows _Gpr48_−/− have smaller lens compared with wild-type mice. (d) The ratio of soluble and insoluble αA-crystallin has been changed in _Gpr48_−/− mice with significant increase of insoluble αA-crystallin in _Gpr48_−/− mice. (e) Activation of αA-crystallin promoter reporter by Gpr48 active mutant T755I. (f) Ganglion cells loss and thinning of inner and outer nuclear layers in _Gpr48_−/− mice at 6 months. [Scale bars: 340 μm (b) and 100 μm (f).]

Fig. 3.

Fig. 3.

Expression changes of key transcription factors in _Gpr48_−/− mouse eyes. (a) Real-time PCR analysis of transcription factors involved in ocular anterior development shows that Pitx2 is down-regulated in P0 _Gpr48_−/− mice. The expression levels of Foxc1, Foxc2, Foxe3, Lmx1b1, and Pax6 are not changed. (b) The expression levels of transcription factors, including Pitx2, Foxc1, Foxc2, Foxe3, Lmx1b1, and Pax6, were confirmed by RT-PCR, showing decreased expression of Pitx2 in the E12.5 _Gpr48_−/− embryo. (c) Decreased expression of Pitx2 protein using Western blot analysis with the E12.5-day embryo in _Gpr48_−/− mice. (d) Whole-mount in situ hybridization (Pitx2 and Foxc1) and immunofluorescence staining (Pax6) in the E12.5 embryo demonstrates that Pitx2, but not Foxc1 and Pax6, was significantly decreased in the _Gpr48_−/− embryo. [Scale bars: 85 μm (b).]

Fig. 4.

Fig. 4.

Regulation of Pitx2 expression by Gpr48 through cAMP-PKA-CREB signaling pathway. (a) Activation of cAMP production by constitutively active Gpr48 mutant (T755I). (b) Activation of CRE luciferase reporter activity by Gpr48 T755I mutant receptor. (c) Diagram of the CRE-binding site in the Pitx2 promoter and two Pitx2 luciferase reporters: luc-108, which contains the CRE site, and luc-67, which does not. (d) Activation of Pitx2 108-luc reporter by Gpr48 active mutant (T755I). Pitx2 67-luc reporter (no CRE-binding site) was not activated by the active mutant receptor. (e) Point mutation of the CRE site (from TGACGTCA to TGAAATCA) of the Pitx2 promoter dramatically decreased the activation by Gpr48 active receptor (T755I). (f and g) PKI and H-89 inhibit the activation of the Pitx2 108-luc reporter mediated by the Gpr48 active receptor, respectively. (h) Binding of CREB transcription factor to the CRE-binding site of the Pitx2 promoter region by EMSA assays. Lane 1, free probe; lane 2, nuclear extract from αT3–1 cells showing strong binding with Pitx2 CRE probe; lane 3, 200-fold cold Pitx2 CRE probe successfully shifted the binding; lane 4, 200-fold mutant Pitx2 CRE probe failed to shift the band; lane 5, anti-IgG antibody did not shift the binding of the CRE probe; lane 6, anti-CREB antibody successfully shifted the binding complex of the Pitx2 CRE probe. (i) ChIP assays of the CRE site in the Pitx2 promoter demonstrate that anti-CREB antibody can successfully precipitate the DNA fragment in the promoter region of Pitx2. (j) Diagram of Gpr48 signaling pathways and regulation of ASD by Gpr48 through down-regulation of Pitx2 expression. Activation of Gpr48 leads to the production of intracellular cAMP and the activation of PKA. PKA can phosphorylate and regulate the activity of CREB, a key transcription factor that regulates the expression levels of many key genes, such as Pitx2 and α_A-crystallin_, in anterior segment development and dysgenesis.

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