Dai G, Imagawa W, Liu B, Szpirer C, Levan G, Kwok S C, Soares M J
Department of Physiology, University of Kansas Medical Center, Kansas City 66160, USA.
Endocrinology. 1996 Nov;137(11):5020-7. doi: 10.1210/endo.137.11.8895376.
In this report, we have investigated placental lactogens (placental lactogen-I, PL-I; PL-I variant, PL-Iv; PL-II) expressed by differentiated Rcho-1 trophoblast cells. A complementary DNA (cDNA) library to differentiated Rcho-1 trophoblast cells was constructed and screened with probes to detect PL-I and PL-II. Sequence analysis of three independent Rcho-1 PL-I cDNAs indicated that they significantly differed from the previously reported PL-I sequence but more closely resembled a related cDNA referred to as PL-I mosaic (PL-Im). Upon further analysis, Rcho-1 PL-I/PL-Im transcripts could be detected in Rcho-1 trophoblast cells and normal developing placental tissue; however, the previously reported PL-I transcript could not be identified from the same sources. Given these results, we examined the original PL-I cDNA by PCR and nucleotide sequence analyses. The sequence differed from the original report and was found to be identical to the Rcho-1 PL-I and PL-Im cDNA clones. Thus, PL-I, Rcho-1 PL-I, and PL-Im are equivalent and should be referred to as PL-I. The PL-I gene was localized to chromosome 17 of the rat genome, similar to other PRL family members. Rcho-1 PL-II cDNAs were identical to the published PL-II sequence. PL-Iv cDNAs were isolated from differentiated Rcho-1 cells via an RT-PCR strategy and found to be identical to previously isolated PL-Iv cDNAs. Rcho-1 PL-I and PL-II cDNAs were subcloned into the pcDNA3 expression vector and recombinant protein produced in HRP-1 cells. Both recombinant Rcho-1 PL-I and PL-II proteins significantly stimulated the proliferation of lactogen-dependent rat Nb2 lymphoma cells and mouse mammary epithelial cells. In summary, we show that the Rcho-1 PL-I corresponds to PL-Im and Rcho-1 PL-Iv and PL-II are identical to their previously described placental counterparts. Additionally, both recombinant Rcho-1 PL-I and PL-II proteins are biologically active.
在本报告中，我们研究了分化的Rcho-1滋养层细胞所表达的胎盘催乳素（胎盘催乳素-I，PL-I；PL-I变体，PL-Iv；PL-II）。构建了一个针对分化的Rcho-1滋养层细胞的互补DNA（cDNA）文库，并用探针进行筛选以检测PL-I和PL-II。对三个独立的Rcho-1 PL-I cDNA进行序列分析表明，它们与先前报道的PL-I序列有显著差异，但更类似于一种相关的cDNA，称为PL-I嵌合体（PL-Im）。进一步分析发现，Rcho-1 PL-I/PL-Im转录本可在Rcho-1滋养层细胞和正常发育的胎盘组织中检测到；然而，从相同来源无法鉴定出先前报道的PL-I转录本。鉴于这些结果，我们通过PCR和核苷酸序列分析检查了原始的PL-I cDNA。该序列与原始报告不同，并且被发现与Rcho-1 PL-I和PL-Im cDNA克隆相同。因此，PL-I、Rcho-1 PL-I和PL-Im是等同的，应称为PL-I。PL-I基因定位于大鼠基因组的17号染色体，与其他催乳素（PRL）家族成员相似。Rcho-1 PL-II cDNA与已发表的PL-II序列相同。通过逆转录聚合酶链反应（RT-PCR）策略从分化的Rcho-1细胞中分离出PL-Iv cDNA，发现其与先前分离的PL-Iv cDNA相同。将Rcho-1 PL-I和PL-II cDNA亚克隆到pcDNA3表达载体中，并在HRP-1细胞中产生重组蛋白。重组的Rcho-1 PL-I和PL-II蛋白均显著刺激了依赖催乳素的大鼠Nb2淋巴瘤细胞和小鼠乳腺上皮细胞的增殖。总之，我们表明Rcho-1 PL-I与PL-Im相对应，并且Rcho-1 PL-Iv和PL-II与其先前描述的胎盘对应物相同。此外，重组的Rcho-1 PL-I和PL-II蛋白均具有生物活性。
