Expression Of CREG Protein In Mice Embryogenesis

Objective The cellular repressor of E1A stimulated genes (CREG), as a noval transcriptional regulators, was cloned originally from a HeLa cDNA library. It can antagonize the E2F-dependent transcriptional activation and the oncogenic transformation of primary cells induced by the Adenovirus E1A oncoprotein cooperating with the oncogene ras. Opposite to the effects of E1A, the physiological function of CREG can be to inhibit cell growth and/or promote differentiation.Although expressed at extremely low levels in undifferentiated embryonic stem cells, CREG mRNA is rapidly induced during differentiation of diverse type cells such as mouse ES, human embryonal carcinoma cells, human leukemia cell line U937 and myeloid progenitor cell line EML cells. It has been reported that CREG is modified by M6P and binding to the insulin-like growth factorⅡ/mannose 6-phosphate receptor is required for CREG-mediated growth suppression in multiple lineages. In 1999, we obtained the high expression of CREG mRNA from the clone of human internal thoracic artery cells-Shenyang (named HITASY cells) in the differentiated phenotype using mRNA differential display PCR technology. Further research found that both mRNA and protein of CREG were synchronously increased in HITASY cells when they undergone the phenotypic change from the dedifferentiated state to the differentiated one induced by serum withdrawal. Subsequent research indicated that overexpression of CREG promoted differentiation of primary rat vascular smooth muscle cells (VSMCs) by binding together with serum response factor to the CArG site of smooth muscleα-actin (SMα-actin) promoter. To gain greater insight into the effect of CREG on the underlying phenotypic modulation in VSMCs, our lab has primarily focused on the regulation role of CREG during neointimal proliferation and the study results showed the oppositive relationships between the cells proliferation index and CREG mRNA expression in the arterial wall at different time point after balloon injury, and increased expression of CREG protein was associated with increased SMα-actin expression. All of the previous studies illuminated that CREG was involved in the process of VSMCs differentiation and phenotype modulation both in vitro and in vivo.It was previously found that CREG was widely expressed in adult tissues such as brain, heart, lung, liver, intestine, and kidney of mice. To further clarify the relationship between the expression of CREG and cellular differentiation, in this study, we used immunohistochemistry, Western blot and RT-PCR methods to detect the expression of CREG in mice embryogenesis and the distribution of the expression in 18.5-dpc (days post coitus) embryos. We also observed the relationship between CREG and vasculogenesis in order to provide necessary evidence for studying function of CREG in the future.Methods①The model of mouse embryo was established by injection PMSG and hCG into female mice, then putting them into the cage of male mice for mating. The pregnant mice were killed at different time after the day of post coitus and embryo mice were obtained. The samples were imbedded in paraffin and then were cut into sections, stained with HE, finally observed by microscopy.②Using immunohistochemistry, we detected the expression and distribution of CREG in embryo mice of different stage.③Western blot was used to detect the expression of CREG in mice embryogenesis and in 18.5-dpc embryos.④Total RNA was extracted by Trizol from embryo mice. Reverse transcriptase-PCR was used to measure the expression change of CREG mRNA at different stage in embryo mice.⑤Sections were stained to observe the morphology change of vasculogenesis in embryo, new born and adult mice.⑥The relationship between SMα-actin and CREG was detected in vasculogenesis at different stage.⑦The expression of CREG was also observed in vessels of different adult organs.Results①Expression of CREG protein in different stage mouse embryo: Immunohistochemistry analysis at different stage of mouse embryos showed that the earliest time point of CREG expression was the day of 5.5 pc, which was located in primitive ectoderm. Neither inner cell mass nor trophectoderm was found CREG protein at day 4.5 embryos. At day of 6.5 and 9.5 development, CREG protein was found most widely expressed in endoderm, mesoderm and ectoderm. When mouse development entered into the phase of organogenesis and histogenesis, CREG protein was distributed in different organs with different initated time point. At day of 13.5 and 15.5 pc, CREG protein was detected in heart, brain, and liver, but not in lung, intestine and kidney. Up to the day of 18.5 pc, the expression of CREG was observed in all above organs. CREG was expressed almost in all stages of embryos. Western blot and RT-PCR analysis identified that the expression of CREG was gradually increased and reached to the highest in the 18.5-dpc embryos.②The distribution of CREG protein in organs of 18.5-dpc embryos: Total protein abstracted from different organs of 18.5-dpc embryos was analyzed by Western blot. The result showed that the CREG protein was expressed at the higher level in brain, heart, intestine and kidney in 18.5-dpc embryos than that in lung and liver. By immunohistochemistry, we observed that CREG protein was located in colloid cells, myocardial cells, alveolar epithelial cells, hepatic cells, intestinal mucosa cells and renal tubule epithelia cells which were dark brown homogeneous in cytoplasm, but not in nuclus.③Morphology characteristic of vessels in embryogenesis mice and the location of CREG protein: In 9.5-dpc embryos, vessel wall was composed of one-layer endothelial cells. The gap of cells was large and blood cells were located in vascular lumens. Immunohistochemistry showed that CREG was expressed (++) only in the endothelial cells associated with the initial formation of vascular lumens, SMα-actin was not detected. In 10.5-dpc embryos, vessels are surrounded with two-layer or multi-layer cells which were arranged loosely. Both the expressions of SMα-actin and CREG protein were found (+) with co-localization in adventitial cells in 10.5-dpc embryos vessels. In 12.5-dpc embryo, much more the VSMCs are emerged surrounding the vessel. With the differentiation and recruitment of VSMCs into the embryonic vascular structures, SMα-actin protein (++) expressed to increase in VSMCs. Besides in the endothelial cells and VSMCs, CREG protein was also detected in the adventitial cells (++). The expressions of SMα-actin and CREG proteins were much stronger in 12.5-dpc than that in 10.5-dpc. From 15.5-dpc to 18.5-dpc, endomembrane, tunica media and adventitia are much clearer and lumens are much larger than ever with vessel development. Meanwhile, SMα-actin protein was expressed (+++) either in 15.5-dpc or in 18.5-dpc. However, compared to the SMα-actin protein, CREG expression which reached to maximum in 15.5-dpc embryos vessel (+++), was found to reduce slightly (++) and keep on from 18.5-dpc embryos vessel to adults ones.④Morphous change of vessels and expression of CREG in new born and adult mice: In postnatal 1 d mouse, vessel structure composed of endomembrane, tunica media and adventitia is clear and lumen is large. Different layer cells of vessels are arranged tightly and separated with other tissue distinctly. VSMCs in media layer are long and thin, nucelus of which are large. Along with vascular development, aorta of postnatal 28 d mouse is more mature. Extracellular matrix is increased and VSMCs are divided by elastic fibers and collagen fibers. Nucelus of VSMCs are smaller than ever and the ratio of nucleus to cytoplasm is decreased. SMα-actin and CREG proteins were expressed (++) in vascular wall of postnatal 1, 28 d and 2m mouse.⑤Expression of CREG in vessels of different organs: The CREG protein expression with different quantity was identified to lie not only in large vessel but also in micro vessel of different organs such as heart, lungs, spleen and kidneys in the adult mice. The expression of CREG proteins were much stronger in coronary (+++) than that in lung, spleen and kidney (+).Conclusion The earliest time point of CREG expression was in the 5.5-dpc embryos. The level of CREG was gradually increased and the highest in the 18.5-dpc. The distribution of CREG in organs in 18.5-dpc embryos was similar to that in adult mice. These results suggest that CREG was expressed in mice embryogenesis and probably participate in differentiation of these organs. In 9.5-dpc embryos, CREG was expressed only in the endothelial cells associated with the initial formation of vascular lumens. With the differentiation and recruitment of the VSMCs into the embryonic vascular structures, the expressions of both SMα-actin and CREG were found to increase gradually from 10.5-dpc embryos to adult vessels. These results provide the evidence for the suggestion that CREG might play an important role in regulation of murine vasculogenesis by inducing and holding the differentiation of vascular cells, especially in VSMCs.