Takahashi Yoko, Maki Takakuni, Liang Anna C, Itoh Kanako, Lok Josephine, Osumi Noriko, Arai Ken
Department of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine, Tohoku University School of Medicine, Sendai, Japan; Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA.
Brain Res. 2014 Dec 17;1593:1-8. doi: 10.1016/j.brainres.2014.10.029. Epub 2014 Oct 22.
Pericytes are vascular mural cells embedded within the basal lamina of blood micro-vessels. Within the neurovascular unit, pericytes play important roles in regulating neurovascular homeostasis by secreting soluble factors, such as matrix metalloproteinases (MMPs). However, little is known about the regulatory signaling pathways in brain pericytes. Here we show that transforming growth factor-β1 (TGF-β1) induces MMP-9 upregulation in pericytes via p38 mitogen-activated protein (MAP) kinase signaling. Cultured human brain vascular pericytes were used in this study. When the brain pericytes were treated with purified human TGF-β1 (0.1-10ng/mL for 24h), the levels of MMP-2 and MMP-9 in culture media were significantly increased in a concentration dependent manner as measured by gelatin zymography. WST assay confirmed that TGF-β1 did not affect cell survival of the brain pericytes. A TGF-β-receptor inhibitor SB431542 (0.5-5μM) decreased the TGF-β1-induced upregulation of MMP-2 and MMP-9. To assess the underlying intracellular mechanisms, we focused on p38 MAP kinase signaling, which is one of the major downstream kinases for TGF-β1. A well-validated p38 MAP kinase inhibitor SB203580 (0.5-5μM) cancelled the effect of TGF-β1 in upregulation of MMP-9 but not MMP-2. Western blotting confirmed that TGF-β1 treatment increased the level of p38 MAP kinase phosphorylation in pericytes, and again, the TGF-β-receptor inhibitor SB431542 (0.5-5μM) blocked the TGF-β1-induced phosphorylation of p38 MAP kinase. Both TGF-β1 and MMP-9 are major neurovascular mediators, and therefore, our current finding may suggest a novel mechanism for how pericytes regulate neurovascular homeostasis.
周细胞是嵌入在血液微血管基膜内的血管壁细胞。在神经血管单元中，周细胞通过分泌可溶性因子，如基质金属蛋白酶（MMPs），在调节神经血管稳态中发挥重要作用。然而，关于脑周细胞中的调节信号通路知之甚少。在此，我们表明转化生长因子-β1（TGF-β1）通过p38丝裂原活化蛋白（MAP）激酶信号通路诱导周细胞中MMP-9上调。本研究使用培养的人脑血管周细胞。当用脑周细胞与纯化的人TGF-β1（0.1 - 10 ng/mL，处理24小时）处理时，通过明胶酶谱法测定，培养基中MMP-2和MMP-9的水平以浓度依赖性方式显著增加。WST检测证实TGF-β1不影响脑周细胞的细胞存活。TGF-β受体抑制剂SB431542（0.5 - 5 μM）可降低TGF-β1诱导的MMP-2和MMP-9上调。为了评估潜在的细胞内机制，我们聚焦于p38 MAP激酶信号通路，它是TGF-β1的主要下游激酶之一。一种经过充分验证的p38 MAP激酶抑制剂SB203580（0.5 - 5 μM）消除了TGF-β1对MMP-9上调的作用，但对MMP-2没有作用。蛋白质印迹法证实TGF-β1处理增加了周细胞中p38 MAP激酶的磷酸化水平，并且，TGF-β受体抑制剂SB431542（0.5 - 5 μM）再次阻断了TGF-β1诱导的p38 MAP激酶磷酸化。TGF-β1和MMP-9都是主要的神经血管介质，因此，我们目前的发现可能提示了周细胞调节神经血管稳态的一种新机制。
