Cecile Rouleau1, Kunyuan Cui, Laurie Feldman. 1. Laboratory for Cell and Molecular Biology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass 02215, USA.
Abstract
OBJECTIVE: We hypothesized that thrombopoietin (TPO) exerts its mitogenic effects on erythroid cells, at least in part, via an interaction of TPO with the cells' erythropoietin receptor (EPO-R). METHODS: We used BaF3 cells stably transfected with EPO-R to demonstrate that TPO alone is sufficient to support the long-term growth and proliferation of BaF3/EPO-R cells and to develop a TPO-dependent variant, BaF3/EPO-R(T), which is highly sensitive to and dependent on TPO for its proliferation. Northern analysis and RT-PCR were used to verify that both BaF3/EPO-R and BaF3/EPO-R(T) cells express EPO-R but lack c-mpl, the TPO receptor. To confirm that TPO responsiveness of BaF3/EPO-R(T) is due to TPO's interaction with EPO-R, EPO-R was downregulated by antisense mRNA. RESULTS: Downregulation of EPO-R in BaF3/EPO-R(T) cells abolishes responsiveness to both EPO and TPO. Viability of EPO-treated transfectants decreased from 95% to 36%, while that of TPO-treated transfectants decreased from 95% to 9% by 48 hours. Nontransfected BaF3/EPO-R(T), and BaF3/EPO-R(T) transfected with vector alone, remained viable and grew in either EPO or TPO. CONCLUSION: Our results suggest a functional EPO-R may be necessary and sufficient for TPO to exert its mitogenic effects on erythroid cells.
OBJECTIVE: We hypothesized that thrombopoietin (TPO) exerts its mitogenic effects on erythroid cells, at least in part, via an interaction of TPO with the cells' erythropoietin receptor (EPO-R). METHODS: We used BaF3 cells stably transfected with EPO-R to demonstrate that TPO alone is sufficient to support the long-term growth and proliferation of BaF3/EPO-R cells and to develop a TPO-dependent variant, BaF3/EPO-R(T), which is highly sensitive to and dependent on TPO for its proliferation. Northern analysis and RT-PCR were used to verify that both BaF3/EPO-R and BaF3/EPO-R(T) cells express EPO-R but lack c-mpl, the TPO receptor. To confirm that TPO responsiveness of BaF3/EPO-R(T) is due to TPO's interaction with EPO-R, EPO-R was downregulated by antisense mRNA. RESULTS: Downregulation of EPO-R in BaF3/EPO-R(T) cells abolishes responsiveness to both EPO and TPO. Viability of EPO-treated transfectants decreased from 95% to 36%, while that of TPO-treated transfectants decreased from 95% to 9% by 48 hours. Nontransfected BaF3/EPO-R(T), and BaF3/EPO-R(T) transfected with vector alone, remained viable and grew in either EPO or TPO. CONCLUSION: Our results suggest a functional EPO-R may be necessary and sufficient for TPO to exert its mitogenic effects on erythroid cells.
Authors: Nina Hahn; Luca Büschgens; Nicola Schwedhelm-Domeyer; Sarah Bank; Bart R H Geurten; Pia Neugebauer; Bita Massih; Martin C Göpfert; Ralf Heinrich Journal: Front Mol Neurosci Date: 2019-10-11 Impact factor: 5.639
Authors: John E Baker; Jidong Su; Anna Hsu; Yang Shi; Ming Zhao; Jennifer L Strande; Xiangping Fu; Hao Xu; Annie Eis; Richard Komorowski; Eric S Jensen; James S Tweddell; Parvaneh Rafiee; Garrett J Gross Journal: Cardiovasc Res Date: 2007-09-22 Impact factor: 10.787