Jullien Nicolas, Roche Catherine, Brue Thierry, Figarella-Branger Dominique, Graillon Thomas, Barlier Anne, Herman Jean-Paul
Aix-Marseille Université, CNRS, UMR7286, 13015 Marseille, France.
Aix-Marseille Université, CNRS, UMR7286, 13015 Marseille, France; Laboratory of Molecular Biology, APHM Conception, 13385 Marseille, France.
PLoS One. 2015 Mar 30;10(3):e0120010. doi: 10.1371/journal.pone.0120010. eCollection 2015.
To test the role of wtPIT-1 (PITWT) or PIT-1 (R271W) (PIT271) in somatolactotroph cells, we established, using inducible lentiviral vectors, sublines of GH4C1 somatotroph cells that allow the blockade of the expression of endogenous PIT-1 and/or the expression of PITWT or PIT271, a dominant negative mutant of PIT-1 responsible for Combined Pituitary Hormone Deficiency in patients. Blocking expression of endogenous PIT-1 induced a marked decrease of cell proliferation. Overexpressing PITWT twofold led also to a dose-dependent decrease of cell proliferation that was accompanied by cell death. Expression of PIT271 induced a strong dose-dependent decrease of cell proliferation accompanied by a very pronounced cell death. These actions of PIT271 are independent of its interaction/competition with endogenous PIT-1, as they were unchanged when expression of endogenous PIT-1 was blocked. All these actions are specific for somatolactotroph cells, and could not be observed in heterologous cells. Cell death induced by PITWT or by PIT271 was accompanied by DNA fragmentation, but was not inhibited by inhibitors of caspases, autophagy or necrosis, suggesting that this cell death is a caspase-independent apoptosis. Altogether, our results indicate that under normal conditions PIT-1 is important for the maintenance of cell proliferation, while when expressed at supra-normal levels it induces cell death. Through this dual action, PIT-1 may play a role in the expansion/regression cycles of pituitary lactotroph population during and after lactation. Our results also demonstrate that the so-called "dominant-negative" action of PIT271 is independent of its competition with PIT-1 or a blockade of the actions of the latter, and are actions specific to this mutant variant of PIT-1.
为了测试野生型PIT-1（PITWT）或PIT-1（R271W）（PIT271）在生长催乳素细胞中的作用，我们使用诱导型慢病毒载体建立了GH4C1生长激素细胞亚系，该亚系能够阻断内源性PIT-1的表达和/或PITWT或PIT271的表达，PIT271是PIT-1的显性负性突变体，可导致患者出现联合垂体激素缺乏症。阻断内源性PIT-1的表达会导致细胞增殖显著减少。PITWT过表达两倍也会导致细胞增殖呈剂量依赖性减少，并伴有细胞死亡。PIT271的表达导致细胞增殖呈强烈的剂量依赖性减少，并伴有非常明显的细胞死亡。PIT271的这些作用与其与内源性PIT-1的相互作用/竞争无关，因为在内源性PIT-1的表达被阻断时这些作用没有改变。所有这些作用都是生长催乳素细胞特有的，在异源细胞中未观察到。PITWT或PIT271诱导的细胞死亡伴有DNA片段化，但不受半胱天冬酶、自噬或坏死抑制剂的抑制，这表明这种细胞死亡是一种不依赖半胱天冬酶的凋亡。总之，我们的结果表明，在正常条件下PIT-1对维持细胞增殖很重要，而当以超正常水平表达时它会诱导细胞死亡。通过这种双重作用，PIT-1可能在哺乳期和哺乳期后垂体催乳素细胞群体的扩增/消退周期中发挥作用。我们的结果还表明，PIT271所谓的“显性负性”作用与其与PIT-1的竞争或后者作用的阻断无关，是PIT-1这种突变变体特有的作用。
