González-Arenas Aliesha, Valadez-Cosmes Paulina, Jiménez-Arellano Carolina, López-Sánchez Mónica, Camacho-Arroyo Ignacio
Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Av. Universidad 3000, Coyoacán 04510, México, D.F., Mexico.
Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Av. Universidad 3000, Coyoacán 04510, México, D.F., Mexico.
J Steroid Biochem Mol Biol. 2014 Oct;144 Pt B:463-70. doi: 10.1016/j.jsbmb.2014.09.007. Epub 2014 Sep 12.
Astrocytomas are the most frequent and aggressive primary brain tumors in humans and constitute the leading cause of brain cancer related deaths. There are reports indicating that progesterone (P4) participates in the growth of astrocytomas through the interaction with its intracellular receptor (PR). Recently, it has been found that P4 induces the growth of several tumors through the up-regulation of progesterone-induced blocking factor (PIBF), a protein that has been related to the immunologic and proliferative actions of P4. U373 cells derived from a human astrocytoma grade III were used to study the role of P4 in PIBF expression and the effects of the latter in cell number. By using RT-PCR and Western blot techniques, we found that U373 cells express PIBF mRNA and protein. P4 (10nM and 100nM) increased PIBF mRNA expression after 1 and 3h of treatment, respectively, and this increase lasted 24h. This effect was blocked by the PR antagonist, RU486. Two PIBF isoforms were detected: one of 57kDa and the predominant one of 90kDa. The content of the 90kDa isoform increased after 12h of P4 treatment, and RU486 also blocked this increase. We observed that PIBF was released into the extracellular medium, being the 57kDa isoform the most abundant in this compartment. Immunofluorescence analysis showed that PIBF was localized in both the cytoplasm and nucleus. The effects of PIBF on cell number were analyzed for five consecutive days. PIBF (200ng/mL) significantly increased the number of U373 cells on days 2-5. Co-immunoprecipitation and Western blot assays revealed that PIBF associates to IL-4 receptor, and increases JAK1 and STAT6 phosphorylation at 20min. Our results suggest that P4 regulates PIBF expression in U373 cells through PR, and that PIBF increases cell number through IL-4 receptor/JAK1/STAT6 signaling pathway.
星形细胞瘤是人类最常见且侵袭性最强的原发性脑肿瘤，也是脑癌相关死亡的主要原因。有报道表明，孕酮（P4）通过与其细胞内受体（PR）相互作用参与星形细胞瘤的生长。最近发现，P4通过上调孕酮诱导阻断因子（PIBF）来诱导多种肿瘤生长，PIBF是一种与P4的免疫和增殖作用相关的蛋白质。源自人III级星形细胞瘤的U373细胞用于研究P4在PIBF表达中的作用以及PIBF对细胞数量的影响。通过使用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹技术，我们发现U373细胞表达PIBF信使核糖核酸（mRNA）和蛋白质。P4（10纳摩尔和100纳摩尔）分别在处理1小时和3小时后增加PIBF mRNA表达，且这种增加持续24小时。这种作用被PR拮抗剂RU486阻断。检测到两种PIBF异构体：一种为57千道尔顿，主要的一种为90千道尔顿。P4处理12小时后，90千道尔顿异构体的含量增加，RU486也阻断了这种增加。我们观察到PIBF被释放到细胞外培养基中，57千道尔顿异构体在该部分最为丰富。免疫荧光分析表明，PIBF定位于细胞质和细胞核中。连续五天分析了PIBF对细胞数量的影响。PIBF（200纳克/毫升）在第2至5天显著增加U373细胞数量。免疫共沉淀和蛋白质免疫印迹分析显示，PIBF与白细胞介素-4受体结合，并在20分钟时增加Janus激酶1（JAK1）和信号转导子和转录激活子6（STAT6）的磷酸化。我们的结果表明，P4通过PR调节U373细胞中PIBF的表达，并且PIBF通过白细胞介素-4受体/JAK1/STAT6信号通路增加细胞数量。
