FUNCTIONAR ROFE OF C-FOS IN DIFFERENTIATION AND PROLIFERATION OF MATURE B CELLS

C-Fos诱导生发中心B细胞凋亡我们利用携带c-Fos基因的转基因小鼠，在干扰素-a/b诱导的Mx启动子(Mx-c-FOS)或H-2^b启动子(H2-c-FOS)的控制下，研究了c-Fos在成熟B细胞分化为产生免疫球蛋白的细胞中的作用。Mx-c-fos小鼠脾B细胞与脂多糖和重组人白细胞介素4共同培养，培养3d后即可形成IgG1~+B细胞。从d~2加入Ien-a/b后，24 h内IgG1~+B细胞发育受阻，凋亡细胞增多，提示c-Fos可诱导Ig类转换型B细胞凋亡。为了证实c-Fos在体内对B细胞分化的影响，用DNP-OVA免疫H2-c-FOS小鼠，小鼠在血清中产生原代IgM而不产生Ig G抗DNP抗体，也不能在脾中产生生发中心。通过将H2-c-fos小鼠与携带bcl…的转基因小鼠交配，挽救了生发中心形成的扰动。含免疫球蛋白启动子的More-2基因。但在双转基因小鼠中，原代IgG1抗DNP抗体的产生仍然受到抑制，提示Bcl2可以延缓c-Fos诱导的Ig类转换B细胞的凋亡时间，但不能挽救死亡。由于c-Fos在AGS作用的成熟B细胞中被诱导，并且生发中心自身反应性B细胞的克隆性删除对Bcl2不敏感，这些结果表明c-Fos在生发中心B细胞的克隆性删除中起因果作用。c-fos的延长表达抑制了休眠的造血干细胞的细胞周期进入。为了研究c-fos在造血干细胞中的作用，我们使用了在干扰素-a/b诱导型Mx-启动子(Mx-c-fos)控制下携带c-fos基因的转基因小鼠的骨髓(BM)细胞和c-fos缺陷小鼠的胎肝细胞。C-fos在LIN^-SCA-1^+BM细胞中的长时间表达通过将其保持在细胞周期的G0/G1期来抑制因子依赖的集落形成和基质细胞层的造血。当外源性c-fos下调时，这些间质层上的LIN^-SCA-1^+BM细胞进入细胞周期。但异位c-fos不影响LIN^-SCA-1^+BM细胞进入细胞周期后的集落形成。此外，c-fos基因缺陷小鼠胎肝细胞的因子依赖性和基质依赖性造血细胞并未受到损害，内源性c-fos对造血干细胞的细胞周期进程不是必需的。这些结果表明，在原始造血干细胞中诱导的c-fos负向控制细胞周期的进展，并将其维持在休眠状态。较少

c-Fos Induces Apoptosis in Germinal Center B CellsWe examined a role of c-Fos in differentiation of mature B cells into IgG producing cells using transgenic mice carrying the c-fos gene under the control of the IFN-a/b-inducible Mx promoter (Mx-c-fos) or the constitutive H-2^b promoter (H2-c-fos). Splenic B cells from Mx-c-fos mice were cultured with LPS and rlL-4, and IgG1^+ B cells were developed in the culture after d 3. When IEN-a/b was added in the culture from d 2, development of IgG1^+ B cells was perturbed and the number of apoptotic cells increased within 24 h, suggesting that c-Fos induces apoptosis in Ig class-switching B cells. To confirm the effect of c-Fos on the B cell differentiation in vivo, H2-c-fos mice were immunized with DNP-OVA.The mice produced primary IgM but not IgG anti-DNP Ab in sera and failed to generate germinal centers in spleen. The perturbation of germinal center formation in H2-c-fos mice was rescued by mating them with transgenic mice carrying the bcl … More -2 gene with the Ig promoter. However, primary IgG1 anti-DNP Ab production was still suppressed in doubly transgenic mice, suggesting that Bcl-2 can delay time of c-Fos-induced apoptosis in Ig class-switching B cells but cannot rescue the death. Since c-Fos is induced in mature B cells reacted with Ags and clonal deletion of self-reactive B cells in germinal centers is insensitive to Bcl-2, these results suggest that c-Fos plays a causal role in clonal deletion of germinal center B cells.Prolonged Expression of c-fos Suppresses Cell Cycle Entry of Dormant Hematopoietic Stem CellsThe proto-oncogene c-fos was transiently up-regulated in primitive hematopoietic stem (Lin^-Sca-1^+) cells stimulated with stem cell factor, interleukin-3, and interleukin-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-a/b inducible Mx-promoter (Mx-c-fos), and fetal liver cells from c-fos-deficient mice. Prolonged expression of the c-fos in Lin^- Sca-1^+ BM cells inhibited factor dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin^- Sca-1^+ BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was down-regulated. However, ectopic c-fos did not perturb colony formation by Lin^- Sca-1^+ BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells since the factor dependent and the stroma dependent hematopoiesis by Lin^- Sca-1^+ fetal liver cells from c-fos-deficient mice were not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state. Less