Manzel L, Macfarlane D E
Department of Medicine, Veteran's Administration Medical Center, Iowa City, IA, USA.
Leuk Res. 1997 May;21(5):403-10. doi: 10.1016/s0145-2126(96)00096-3.
Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.
血细胞前体的生长和分化受细胞因子和激素的调节，但其机制尚未完全明确。蛋白激酶C（PKC）同工酶在信号转导通路中被广泛认为具有重要作用。我们已经表明，一种同工酶PKCβ在介导佛波酯诱导的HL-60髓系细胞系生长停滞中具有独特的重要性。1,25-二羟基维生素D3可诱导许多细胞分化并使其生长停滞。它上调PKCβ的表达，增强佛波酯的作用。我们检验了以下假设：使造血细胞生长停滞的细胞因子是通过激活PKCβ来实现的，以及1,25-二羟基维生素D3诱导的分化和生长停滞是由PKCβ同工酶基因表达上调所致。测定了五种细胞因子（肿瘤坏死因子α、转化生长因子β1、γ-干扰素、白细胞介素-1和粒细胞集落刺激因子）与1,25-二羟基维生素D3的组合对十种人白血病细胞系（THP-1、HL-60 S、HL-60 PET、U937、K562、Jurkat、MOLT-4、RPM1 8402、KG-1和KG-1a）生长的影响。四种细胞系（THP-1、HL-60 S和PET以及U937）在与1,25-二羟基维生素D3接着与转化生长因子β1共同孵育时出现完全生长停滞。测定了每种细胞系在与1,25-二羟基维生素D3孵育24或48小时前后编码PKCα、β和δ的mRNA的表达情况。对转化生长因子β1敏感的细胞系各自内源性表达PKCδ，或者其表达随1,25-二羟基维生素D3而上调。U937细胞中PKCα表达不足，HL-60 PET细胞中PKCβ表达不足。这些数据表明PKCδ可能负责介导转化生长因子β1引起的生长停滞。为了直接检验这一假设，我们在加入1,25-二羟基维生素D3和转化生长因子β1期间用两种双吲哚马来酰亚胺PKC抑制剂处理细胞。令人惊讶的是，PKC抑制剂并未阻断1,25-二羟基维生素D3和转化生长因子β1诱导的生长停滞。该实验有力地表明，转化生长因子β1诱导的生长停滞以及1,25-二羟基维生素D3对这种生长停滞的增强作用均不是由可被双吲哚马来酰亚胺抑制的PKC同工酶介导的。
