Stromal cells in the bone marrow provide an optimal microenvironment for hematopoiesis through cell-to-cell interaction as well as cytokine production. We have previously established 33 bone marrow stromal cell lines from SV40 T-antigen transgenic mice, and demonstrated the lineage-specific activity of supporting hematopoiesis (

J Cell Physiol
1995
:
164
;
55
). Of the 33 cell lines, 27 clones showed stimulatory abilities to support large erythroid colony formation in the presence of erythropoietin, while 6 cell lines did not. Since these activities were not dependent on the amount of produced cytokines, the existence of unknown molecules responsible for the selective activity of erythropoiesis has been implicated. To identify molecules expressed on bone marrow stromal cells which are responsible for determining the stromal cell dependent erythropoiesis, we compared the gene expression profiling by cDNA microarray between cell lines which support erythropoiesis (E+; TBR 9, 184, 31-2) and cell lines which do not (E-; TBR 17, 33, 511). Out of 7226 genes examined, 138 genes were up-regulated 3-fold or more, and 282 genes were down-regulated 3-fold or more, in E+ cell lines. Among these genes, we have selected one of the up-regulated genes, tenascin-C, as a candidate for erythroid progenitor supporting genes, and examined its expression and function in detail. First, we confirmed the expression of tenascin-C by real time quantitative RT-PCR. The expression of tenascin-C adjusted by the expression of GAPDH in the three E+ cell lines were all higher than the three E- cell lines with a mean of 3.6 times. Next, we examined the effect of suppressing tenascin-C expression by small interfering RNA (siRNA) on the erythroid colony formation. siRNAs were synthesized by in vitro transcription method, and were transfected into TBR cell lines by lipofection. At 24 hours after the transfection of tenascin-C siRNA, the expression of tenascin-C was reduced to 10 to 40% of the control siRNA as determined by real time quantitative RT-PCR. The number of erythroid colony dependent on TBR 9 and TBR 184 in the presence of tenascin-C siRNA was significantly lower than that in the presence of control siRNA (13.0±6.5 versus 0.75±0.95 colonies/104 fetal liver cells; p<0.05). These results suggest that tenascin-C plays an important role in the stromal cell dependent erythropoiesis.

Topics:
erythropoiesis, molecule, stromal cells, tenascin, rna, small interfering, cytokine, quantitative real-time polymerase chain reaction, dna, complementary, erythropoietin, gene expression profiling

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2005, The American Society of Hematology
2004
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