Histamine receptor H1 and dermatopontin: new downstream targets of the vitamin D receptor.
In this study, we used multipotential MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. We analyzed co-expressed gene groups to identify new targets for known transcription factor VDR during differentiation. The roles of two genes (histamine receptor H1 and dermatopontin) as downstream targets for the VDR were confirmed by gel electromotility shift, siRNA inhibition, and chromatin immunoprecipitation assays.


BACKGROUND
Osteogenesis is stringently controlled by osteoblast-specific signaling proteins and transcription factors. Mesenchymal stem or multipotential stromal cells from bone marrow (MSCs) have been shown to differentiate into osteoblasts in the presence of vitamin D(3).


METHODS
We used MSCs and microarray assays to follow the changing patterns of gene expression as MSCs were differentiated to osteoblasts. The data were analyzed with a previously developed strategy to identify new downstream targets of the vitamin D receptor (VDR), known osteogenesis transcription factor. Hierarchical clustering of the data identified 15 distinct patterns of gene expression. Three genes were selected that expressed in the same time-dependent pattern as osteocalcin, a known target for the VDR: histamine receptor H1 (HRH1), Spondin 2 (SPN), and dermatopontin (DPT). RT-PCR, electromotility shift, siRNA inhibition assays, and chromatin immunoprecipitation assays were used to analyze the role of VDR in activation of DPT and HRH1 during differentiation.


CONCLUSIONS
RT-PCR assays confirmed that the genes were expressed during differentiation of MSCs. The roles of two genes as downstream targets for the VDR were confirmed by gel electromotility shift and chromatin immunoprecipitation assays that showed the presence of VDR complex binding sequences. Overexpression of VDR in MG-63 osteosarcoma cells induced the expression of HRH1 and DPT. Inhibition studies with siRNA to DPT and HRH1 showed a decrease in MSC differentiation to osteogenic lineage. In addition, osteogenic differentiation of MSCs was inhibited by the HRH1 inhibitor mepyramine but not the HRH2 inhibitor ranitidine. In conclusion, we show that analysis of co-expressed gene groups is a good tool to identify new targets for known transcription factors.
Pochampally
Radhika R
RR
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA. rpocham@tulane.edu
Ylostalo
Joni
J
Penfornis
Patrice
P
Matz
Robert R
RR
Smith
Jason R
JR
Prockop
Darwin J
DJ
D001483:Base Sequence; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000502:physiology*; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000502:physiology*; D020869:Gene Expression Profiling; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol / Q000502:physiology*; D011969:Receptors, Histamine H1 / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D011508:Chondroitin Sulfate Proteoglycans; C082019:DPT protein, human; D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D011969:Receptors, Histamine H1