Supplementary Material for: Neuropeptide TLQP-21, a VGF Internal Fragment, Modulates Hormonal Gene Expression and Secretion in GH3 Cell Line

In the present study we demonstrated that TLQP-21, a biologically active peptide derived from the processing of the larger pro-VGF granin, plays a role in mammotrophic cell differentiation. We used an established in vitro model, the GH3 cell line, which upon treatment with epidermal growth factor develops a mammotrophic phenotype consisting of induction of prolactin expression and secretion, and inhibition of growth hormone. Here we determined for the first time that during mammotrophic differentiation, epidermal growth factor also induces Vgf gene expression and increases VGF protein precursor processing and peptide secretion. After this initial observation we set out to determine the specific role of the VGF encoded TLQP-21 peptide on this model. TLQP-21 induced a trophic effect on GH3 cells and increased prolactin expression and its own gene transcription without affecting growth hormone expression. TLQP-21 was also able to induce a significant rise of cytoplasmic calcium, as measured by Fura2AM, due to the release from a thapsigargin-sensitive store. TLQP-21-dependent rise in cytoplasmic calcium was, at least in part, dependent on the activation of phospholipase followed by phosphorylation of PKC and ERK. Taken together, the present results demonstrate that TLQP-21 contributes to differentiation of the GH3 cell line toward a mammotrophic phenotype and suggest that it may exert a neuroendocrine role in vivo on lactotroph cells in the pituitary gland.

本研究证实，TLQP-21——一种由更大分子量的前体VGF粒蛋白（pro-VGF granin）加工生成的生物活性肽——在催乳细胞分化中发挥关键作用。本研究采用已成熟建立的体外模型GH3细胞系：该细胞系经表皮生长因子（epidermal growth factor, EGF）处理后，会形成催乳表型，具体表现为催乳素（prolactin）的表达与分泌被诱导，同时生长激素（growth hormone, GH）的表达被抑制。本研究首次证实，在催乳细胞分化过程中，表皮生长因子还可诱导Vgf基因的表达，并增强VGF蛋白前体的加工与肽类分泌。在获得这一初始发现后，本研究进一步探究了VGF编码的TLQP-21肽在该模型中的具体功能。实验结果显示，TLQP-21可对GH3细胞产生营养支持作用，上调催乳素的表达以及TLQP-21自身的基因转录，且不会对生长激素的表达产生影响。此外，TLQP-21还可诱导胞质钙离子浓度显著升高，该现象可通过Fura2AM检测得到，其机制为钙离子从毒胡萝卜素（thapsigargin）敏感钙库中释放。进一步研究表明，TLQP-21依赖的胞质钙离子浓度升高，至少部分依赖于磷脂酶的激活，随后依次发生蛋白激酶C（protein kinase C, PKC）与细胞外调节蛋白激酶（extracellular signal-regulated kinase, ERK）的磷酸化。综上，本研究结果证实，TLQP-21可促进GH3细胞系向催乳表型分化，且提示其在体内可对垂体腺（pituitary gland）中的催乳素分泌细胞（lactotroph cells）发挥神经内分泌功能。