Mukherjee Subhendu, Sheng Wei, Sun Rui, Janssen Luke J
Firestone Institute for Respiratory Health, St. Joseph's Hospital, Department of Medicine, McMaster University, Hamilton, Ontario, Canada
Firestone Institute for Respiratory Health, St. Joseph's Hospital, Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Am J Physiol Lung Cell Mol Physiol. 2017 Apr 1;312(4):L510-L519. doi: 10.1152/ajplung.00084.2016. Epub 2017 Jan 27.
It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFβ also acts by elevating cytosolic Ca concentration within human pulmonary fibroblasts. Ca/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially generated Ca pulses in the absence of TGFβ, or with TGFβ (1 nM) or vehicle in the presence of various blockers of Ca signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca pulses in the absence of TGFβ increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca signaling and the CamK II pathway significantly reduced TGFβ-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase, or MAP kinase). We also found that TGFβ elevated the expression of CamK IIβ and CamK IIδ, while siRNA silencing of those two subtypes significantly reduced TGFβ-mediated expression of collagen A1 and fibronectin 1. Our data suggest that TGFβ induces the expression of CamK IIβ and CamK IIδ, which in turn are activated by TGFβ-evoked Ca waves in a frequency-dependent manner, leading to increased expression of ECM proteins.
现在已经清楚，除了激活几种复杂的激酶途径（Smad、丝裂原活化蛋白激酶、磷脂酰肌醇-3激酶）外，转化生长因子β（TGFβ）还通过提高人肺成纤维细胞内的胞质钙浓度发挥作用。钙/钙调蛋白依赖性蛋白激酶II（CamK II）也已知可调节成纤维细胞中的基因表达。在本研究中，我们研究了钙信号传导、CamK和其他激酶的激活与细胞外基质（ECM）基因表达之间的相互作用。培养人肺成纤维细胞，并在无TGFβ的情况下用人工产生的钙脉冲刺激，或在存在各种钙信号阻滞剂的情况下用TGFβ（1 nM）或溶剂刺激。分别使用聚合酶链反应（PCR）和蛋白质免疫印迹法来测量基因表达和蛋白质水平。我们发现，在无TGFβ的情况下，钙脉冲以脉冲频率依赖性方式增加ECM基因表达，并且阻断钙信号传导和CamK II途径可显著降低TGFβ介导的ECM基因表达，而对其他激酶途径（Smad、PI3激酶或丝裂原活化蛋白激酶）没有任何影响。我们还发现，TGFβ可提高CamK IIβ和CamK IIδ的表达，而对这两种亚型进行小干扰RNA（siRNA）沉默可显著降低TGFβ介导的胶原蛋白A1和纤连蛋白1的表达。我们的数据表明，TGFβ诱导CamK IIβ和CamK IIδ的表达，这两种亚型又被TGFβ诱发的钙波以频率依赖性方式激活，导致ECM蛋白表达增加。
