Liu Q, Ning W, Dantzer R, Freund G G, Kelley K W
Department of Animal Sciences, University of Illinois, Urbana 61801, USA.
J Immunol. 1998 Feb 1;160(3):1393-401.
Phosphoinositides that are phosphorylated at the D3 position have been reported to activate an atypical, Ca2-independent protein kinase C (PKC) isoform designated PKC-zeta, and overexpression of this enzyme leads to monocytic differentiation. In this study, we cultured human HL-60 promyeloid cells with vitamin D3 and insulin-like growth factor-I (IGF-I), a 70-amino-acid peptide that activates phosphatidylinositol 3'-kinase (PI 3-kinase) in murine promyeloid cells. Two days later, the proportion of cells differentiating into macrophages in serum-free medium, as assessed by expression of the alpha-subunit of the beta2 integrin CD11b, increased from 5 +/- 1% to 25 +/- 3%. Addition of IGF-I increased the proportion of cells differentiating into CD11b-positive macrophages to 78 +/- 5%. In the absence of vitamin D3, IGF-I did not induce expression of CD11b (6 +/- 1%). The IGF-I-promoted macrophage differentiation was blocked specifically by preincubation of HL-60 cells with a mAb (alphaIR3) directed against the IGF type I receptor. Similarly, pretreatment of cells with either alphaIR3 or an IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b expression when cells were cultured with vitamin D3 in serum-containing medium. IGF-I, but not vitamin D3, caused a sevenfold increase in the enzymatic activity of both PI 3-kinase and atypical PKC-zeta. Inhibition of IGF-I-inducible PI 3-kinase with either wortmannin or LY294002 abrogated the IGF-I-induced activation of PKC-zeta and totally blocked the enhancement in macrophage differentiation caused by IGF-I. These data establish that PKC-zeta is a putative downstream target of PI 3-kinase that is activated during IGF-I-promoted macrophage differentiation.
据报道，在D3位置磷酸化的磷酸肌醇可激活一种非典型的、不依赖Ca2+的蛋白激酶C（PKC）亚型，即PKC-ζ，该酶的过表达会导致单核细胞分化。在本研究中，我们用维生素D3和胰岛素样生长因子-I（IGF-I，一种能激活鼠早幼粒细胞中磷脂酰肌醇3'-激酶（PI 3-激酶）的70个氨基酸的肽）培养人HL-60早幼粒细胞。两天后，通过β2整合素CD11b的α亚基表达评估，在无血清培养基中分化为巨噬细胞的细胞比例从5±1%增加到25±3%。添加IGF-I可使分化为CD11b阳性巨噬细胞的细胞比例增加到78±5%。在没有维生素D3的情况下，IGF-I不会诱导CD11b的表达（6±1%）。用针对IGF I型受体的单克隆抗体（αIR3）预孵育HL-60细胞，可特异性阻断IGF-I促进的巨噬细胞分化。同样，当细胞在含血清培养基中用维生素D3培养时，用αIR3或IGF结合蛋白IGFBP-3预处理细胞会导致CD11b表达受到75%的抑制。IGF-I而非维生素D3会使PI 3-激酶和非典型PKC-ζ的酶活性增加7倍。用渥曼青霉素或LY294002抑制IGF-I诱导的PI 3-激酶可消除IGF-I诱导的PKC-ζ激活，并完全阻断IGF-I引起的巨噬细胞分化增强。这些数据表明，PKC-ζ是PI 3-激酶的一个假定下游靶点，在IGF-I促进的巨噬细胞分化过程中被激活。
