Makhijani Nalini S, Bischoff David S, Yamaguchi Dean T
Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, California 90073, USA.
J Cell Physiol. 2005 Jan;202(1):304-13. doi: 10.1002/jcp.20128.
Proliferation of mesenchymal precursors of osteogenic and chondrogenic cells and migration of these precursors to repair sites are important early steps in bone repair. Transforming growth factor-beta (TGF-beta) has been implicated in the promotion of bone repair and may have a role in these processes. Three isoforms of TGF-beta, TGF-beta1, -beta2, and -beta3, are expressed in fracture healing, however, their specific roles in the repair process are unknown. Differential actions of the TGF-beta isoforms on early events of bone repair were explored in the multipotent mesenchymal precursor cell line, C3H10T1/2. Cell migration was determined using a modified Boyden chamber in response to concentrations of each isoform ranging from 10(-12) to 10(-9) g/ml. All three isoforms demonstrated a dose-dependent chemotactic stimulation of untreated C3H10T1/2 cells. Checkerboard assays indicated that all three isoforms also stimulated chemokinesis of the untreated cells. C3H10T1/2 cells treated with all-trans-retinoic acid (ATRA) and expressing relatively higher levels of osteoblastic gene markers such as alkaline phosphatase and collagen type I, lower levels of chondrocytic gene markers collagen type II and aggrecan, and unchanged levels of the adipose marker adipsin did not demonstrate significant chemokinesis or chemotaxis in response to TGF-beta1 or -beta3 at concentrations ranging from 10(-12) to 10(-9) g/ml. In the ATRA-treated cells, TGF-beta2 stimulated a significant increase in chemotaxis only at the highest concentration tested. Cell proliferation was assessed by mitochondrial dehydrogenase activity and cell counts at TGF-beta concentrations from 10(-11) to 10(-8) g/ml. None of the TGF-beta isoforms stimulated cell proliferation in untreated or ATRA-treated C3H10T1/2 cells. Analysis of TGF-beta receptors (TGF-betaR1, -betaR2, and -betaR3) showed a 1.6- to 2.8-fold decrease in mRNA expression of these receptors in ATRA-treated cells.
(1) while all three TGF-beta isoforms stimulate chemotaxis/chemokinesis of multipotent C3H10T1/2 cells, TGF-beta1 and -beta3 do not stimulate chemotaxis in C3H10T1/2 cells treated with ATRA while TGF-beta2 stimulated chemotaxis only at the highest concentration tested. (2) TGF-beta isoforms do not appear to stimulate cell proliferation in C3H10T1/2 cells in either a multipotent state or after ATRA treatment when expressing higher levels of alkaline phosphatase and collagen type I gene markers. (3) Decrease in mRNA expression for TGF-betaR1, -betaR2, and -betaR3 upon ATRA treatment could potentially explain the lack of chemotaxis/chemokinesis in these cells expressing higher levels of alkaline phosphatase and collagen type I.
成骨细胞和软骨生成细胞的间充质前体细胞增殖以及这些前体细胞迁移至修复部位是骨修复早期的重要步骤。转化生长因子-β（TGF-β）被认为可促进骨修复，并且可能在这些过程中发挥作用。TGF-β的三种亚型，即TGF-β1、-β2和-β3，在骨折愈合过程中均有表达，然而它们在修复过程中的具体作用尚不清楚。本研究在多能间充质前体细胞系C3H10T1/2中探讨了TGF-β亚型对骨修复早期事件的不同作用。使用改良的Boyden小室测定细胞迁移情况，检测每种亚型在浓度范围为10^(-12)至10^(-9) g/ml时对细胞的影响。所有三种亚型均对未处理的C3H10T1/2细胞表现出剂量依赖性的趋化刺激作用。棋盘分析表明，所有三种亚型也刺激未处理细胞的趋化运动。用全反式维甲酸（ATRA）处理并表达相对较高水平成骨细胞基因标志物（如碱性磷酸酶和I型胶原）、较低水平软骨细胞基因标志物（II型胶原和聚集蛋白聚糖）且脂肪标志物脂肪酶水平未改变的C3H10T1/2细胞，在10^(-12)至10^(-9) g/ml浓度范围内，对TGF-β1或-β3未表现出明显的趋化运动或趋化性。在经ATRA处理的细胞中，TGF-β2仅在测试的最高浓度下刺激趋化性显著增加。通过线粒体脱氢酶活性和细胞计数评估在TGF-β浓度为10^(-11)至10^(-8) g/ml时的细胞增殖情况。在未处理或经ATRA处理的C3H10T1/2细胞中，没有一种TGF-β亚型刺激细胞增殖。对TGF-β受体（TGF-βR1、-βR2和-βR3）的分析显示，经ATRA处理的细胞中这些受体的mRNA表达下降了1.6至2.8倍。
（1）虽然所有三种TGF-β亚型均刺激多能C3H10T1/2细胞的趋化性/趋化运动，但TGF-β1和-β3在经ATRA处理的C3H10T1/2细胞中不刺激趋化性，而TGF-β2仅在测试的最高浓度下刺激趋化性。（2）当表达较高水平的碱性磷酸酶和I型胶原基因标志物时，TGF-β亚型在多能状态下或经ATRA处理后的C3H10T1/2细胞中似乎不刺激细胞增殖。（3）ATRA处理后TGF-βR1、-βR2和-βR3的mRNA表达下降可能解释了这些表达较高水平碱性磷酸酶和I型胶原的细胞中缺乏趋化性/趋化运动的原因。
