Abstract
We were interested in whether central nervous system progenitor cells possess the signal transduction machinery necessary to mediate cytokine functions and whether this machinery can become activated upon stable expression of a particular cytokine receptor. For this purpose we utilized a previously obtained conditionally immortalized striatum-derived nestin-positive cell line (ST14A). We found that ST14A cells express Jak2, but not Jak1 or Tyk2. An identical pattern of expression was found in embryonic striatal tissue. To evaluate the susceptibility of these cytokine specific cytoplasmic transducers to activation, ST14A cells were stably transfected with the alpha and beta (AIC2A) chains of the murine interleukin-3 receptor. Four independent lines expressing both the alpha and beta receptor subunits were obtained. We found that cells from each of these lines were induced to proliferate upon exposure to interleukin-3. Dose response curve, antibody blocking experiments and binding studies revealed that the response was mediated by the reconstituted high affinity interleukin-3 receptor. Immunoprecipitation studies on these cells showed that Jak2 and Stat5 were being phosphorylated after stimulation of the reconstituted receptor. These results indicate that members of the JAK/STAT family of proteins are expressed in central nervous system progenitor cells and are susceptible to activation through stimulation of an exogenously expressed cytokine receptor, ultimately leading to cell proliferation.
Highlights
In the hematopoietic system, early stem cells generate secondary progenitors which, through successive cell divisions, give rise to the dazzling array of cell types found in blood [1]
Antibody blocking experiments and binding studies revealed that the response was mediated by the reconstituted high affinity interleukin-3 receptor. Immunoprecipitation studies on these cells showed that Jak2 and Stat5 were being phosphorylated after stimulation of the reconstituted receptor. These results indicate that members of the JAK/STAT family of proteins are expressed in central nervous system progenitor cells and are susceptible to activation through stimulation of an exogenously expressed cytokine receptor, leading to cell proliferation
In this study we focussed on obtaining a clonal population of central nervous system (CNS) progenitors in which one cytokine receptor (IL3R) was expressed exogenously, generating a tool in which signal transduction pathways and biological response activated by cytokines in CNS progenitors could be studied
Summary
Cell Culture—ST14A cell line was previously obtained from primary cells dissociated from E14 striatum primordia and immortalized using a retrovirally transduced temperature-sensitive variant (tsA58U19) of SV40 Large T-antigen [20].2 ST14A cells were found to express nestin, a cytoskeletal antigen present in CNS progenitors [22]. Cells were rinsed free of the precipitate, and puromycin at 3 g/ml was applied after a further 48 h. Rinsed cultures were solubilized in NaOH and aliquots assayed for radioactivity by addition of scintillation liquid and for protein by standard protein assays (Bradford) When this experiment was performed in cells exposed to 39 °C, [3H]thymidine of higher specific activity (87 Ci/mmol; Amersham Corp.) was used. The filters were reacted with 4G10 anti-phosphotyrosine (1:1000), anti-Jak (1:1000; UBI, DBA, Italy), or anti-Stat (1:250; Transduction Lab.; DBA, Italy) followed by the respective peroxidase conjugated secondary antibodies and visualized with the ECL detection system (Amersham Corp.) as described by the manufacturer. Membranes of total cell lysates from ST14A and ␣14 cells, as well as those from the embryonic striatum, were reacted with anti-Jak (1:1000; Transduction Lab.; DBA, Italy), anti-Tyk (1:1000; Transduction Lab.; DBA, Italy), and anti-Jak antibodies
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