Bondestam Jonas, Kaivo-oja Noora, Kallio Janne, Groome Nigel, Hydén-Granskog Christel, Fujii Makiko, Moustakas Aristidis, Jalanko Anu, ten Dijke Peter, Ritvos Olli
Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, P O Box 63 (Haartmaninkatu 8), 00014, Helsinki, Finland.
Mol Cell Endocrinol. 2002 Sep 30;195(1-2):79-88. doi: 10.1016/s0303-7207(02)00219-8.
In the mammalian ovary cell growth and differentiation is regulated by several members of the transforming growth factor beta (TGF beta) superfamily including activins, inhibins, growth differentiation factors and bone morphogenetic proteins (BMPs). The effects of TGF beta family members are mediated to the target cells via heteromeric complexes of type I and II serine/threonine kinase receptors which activate Smad signaling protein pathways in various cell types. We have previously shown that inhibin B, a hormonally important product from human granulosa cells, is up regulated by activin and BMPs. Here, we report the use of adenoviral gene transfer methodology to manipulate the TGF beta growth factor signaling system in primary cultures of human granulosa cells. These cells are exceedingly difficult to transfect by conventional transfection methods, but were virtually 100% infected with recombinant adenoviruses expressing green fluorescent protein (GFP). Adenoviruses expressing constitutively active forms of the seven known mammalian type I activin receptor-like kinase receptors (Ad-caALK1 through Ad-caALK7) cause activation of endogenous and adenovirally transferred Smad signaling proteins so that Ad-caALK1/2/3/6 and Ad-caALK4/5/7 induced phosphorylation of the Smad1 and Smad2 pathways, respectively. Activin A and BMP-2 activated the Smad1 and Smad2 pathways as well as inhibin B production as did all the Ad-caALKs. Furthermore, overexpression of adenoviral Smad1 and Smad2 proteins without exogenously added ligands induced inhibin B production. The inhibitory Smad7 protein suppressed BMP-2 and activin induced inhibin B production. Collectively, the present data demonstrate that adenoviral gene transfer provides an effective approach for dissecting the TGF beta signaling pathways in primary ovarian cells in vitro and more specifically indicate that the Smad1 and Smad2 pathways are involved in the regulation of inhibin B production by TGF beta family ligands in the ovary.
在哺乳动物卵巢中，细胞生长和分化受转化生长因子β（TGFβ）超家族的多个成员调控，包括激活素、抑制素、生长分化因子和骨形态发生蛋白（BMP）。TGFβ家族成员的作用通过I型和II型丝氨酸/苏氨酸激酶受体的异源复合物介导至靶细胞，这些受体在各种细胞类型中激活Smad信号蛋白通路。我们之前已表明，抑制素B作为人颗粒细胞的一种重要激素产物，受激活素和BMP上调。在此，我们报告使用腺病毒基因转移方法来操控人颗粒细胞原代培养物中的TGFβ生长因子信号系统。这些细胞极难通过传统转染方法进行转染，但几乎100%被表达绿色荧光蛋白（GFP）的重组腺病毒感染。表达七种已知哺乳动物I型激活素受体样激酶受体组成型活性形式的腺病毒（Ad-caALK1至Ad-caALK7）可激活内源性和腺病毒转移的Smad信号蛋白，因此Ad-caALK1/2/3/6和Ad-caALK4/5/7分别诱导Smad1和Smad2通路的磷酸化。激活素A和BMP-2以及所有Ad-caALK均激活Smad1和Smad2通路以及抑制素B的产生。此外，在无外源添加配体的情况下，腺病毒Smad1和Smad2蛋白的过表达诱导抑制素B的产生。抑制性Smad7蛋白抑制BMP-2和激活素诱导的抑制素B产生。总体而言，目前的数据表明腺病毒基因转移为体外剖析原代卵巢细胞中的TGFβ信号通路提供了一种有效方法，更具体地表明Smad1和Smad2通路参与卵巢中TGFβ家族配体对抑制素B产生的调控。
