RP59 : a novel stem cell protein and mapping of its expression

Abstract

The purpose of this thesis was to study the expression pattern of a novel protein daring developmental processes. Firstly we identified so far unknown mRNA sequences coding for frequently expressed proteins in primitive mesenchymal cells in bone marrow and osteoblasts. Three new sequences were found and one of them, RP59, was selected for further analysis. An artificial RP59 peptide was synthesized in bacteria, which in rum was used for anti-RP59-antibody development in rabbits. With immuno-histochemistry techniques and Western blot presence of protein was demonstrated in primitive mesenchymal cells and osteoblasts at different stages of recruitment. Detection of RP59 protein expression in bone marrow cells was followed up by investigation of yolk sac, fetal liver and spleen, all known to be related to hematopoietic and mesenchymal stem- and progenitor cells. RP59 presence coincided with Flk-1 expression, a cell membrane receptor for VEGF and an established marker for early hematopoiesis and angiogenesis.

In embryonal yolk sac RP59 was present in extra-embryonic blood-islands and the primitive streak and absent from the extra-embryonic endoderm. Later in fetal life, RP59 occurred in hematopoietically active fetal liver, but was depleted from liver after birth. Spleen was colonized by RP59 positive cells in late fetal phase, and these cells persisted up to adult animals in the red pulp of the spleen. In bone marrow, RP59 was detectable starting with its formation. In adult femoral bone marrow, RP59 was easily detected in cells of the erythroid lineage. Megakaryocytes and early osteoblasts. In in vitro cultured ES cells, pluripotent cells from the inner cell mass of mice, RP59 was first absent, but induced in a part of the cells after 7 days of culture. A majority of the RP59+ cells were encapsulated by a primitive endothelial network.

Taken together, we concluded that RP59 is a protein related to primitive mesenchymal cells. During further differentiation, the protein was found downregulated in different ways: in erythrocytes by expulsion of the nucleus, in osteoblasts gradually. In the human genome database, a human homologue to RP59 was located on chromosome 9q22, a region containing breakpoints in hematological and other neoplasms. Because of the strong relation of RP59 to primitive hematopoietic and angiogenic cells, we investigated the presence of human RP59 in pathologies with increased angiogenesis: oral cap~ haemangiomas and pyogenic granulomas. In pyogenic granulomas, RP59 was localized in endothelial cells, pericytes and fibroblasts similar to that in capillary haemangiomas. However, RP59+ polymorphnuclear cells were only observed in pyogenic, granulomas as well as in fibroblasts situated in the intervascular space between the vessels. Together with our earlier findings, this investigation emphasized RP59 expression in cells directly or indirectly originating from the mesodermally derived hemangioblast. When investigating RP59 expression in maxillary bone formation and bone marrow in the rat, surprisingly strong signals were detected in ameloblasts during the secretory phase as well as in enamel.

Immuno-histochemistry at the LM and EM level suggested a pattern of distribution similar to that of amelin. Western blotting of enamel organ proteins, in situ hybridization, RT-PCR and sequence analysis indicated that RP59 was a genuine product of ameloblasts. Comparing the ameloblast expression with the previous results, it seems likely that RP59 can play a role in different biological contexts. We found that RP59 is similar to PEVK-rich proteins, known to serve as a link, with elastic properties, between structural entities in e.g. skeletal muscle, trans-Golgi network and cell nucleus. It is conceivable that a similar Inaction is involved both in enamel matrix assembly as well as intracellularly in progenitor cells.