Roth S, Gong W, Gressner A M
Department of Clinical Chemistry and Central Laboratory, Philipps University, Marburg, Germany.
J Hepatol. 1998 Dec;29(6):915-22. doi: 10.1016/s0168-8278(98)80119-0.
BACKGROUND/AIMS: Kupffer cells (liver resident macrophages) make an important contribution to the perpetuation of liver diseases by synthesis and secretion of TGF-beta. In some cell types TGF-beta, is expressed as a large latent complex containing the latent TGF-beta binding protein (LTBP) in addition to the N-terminal TGF-beta precursor (latency associated peptide). This study aimed to identify LTBP expression in rat Kupffer cells.
Cells were isolated from rat liver by collagenase-pronase reperfusion, purified and cultured under standard conditions. TGF-beta and LTBP expression were characterized using alkaline phosphatase-anti-alkaline phosphatase immunostainings, reverse transcription-polymerase chain reaction and immunoprecipitation of metabolically labeled proteins.
Immunostainings of Kupffer cells with anti-sera against LTBP-1 (ab 39) and LTBP-2 indicated the expression of both LTBP isoforms in addition to the expression of latency associated peptide and TGF-beta. Transcripts of three LTBP isoforms (LTBP-1,-2,-3) and TGF-beta isoforms (TGF-beta-1,-2,-3) were detectable by reverse transcription-polymerase chain reaction. The LTBP-1D splice variant missing a part of the proteinase sensitive hinge region which has recently been described in hepatic stellate cells is expressed in Kupffer cells, too. Metabolic labeling of Kupffer cells with [35S]-Met/Cys followed by immunoprecipitation of the conditioned media using antisera against LTBP-1 and LTBP-2 indicated the secretion of high molecular mass TGF-beta complexes containing LTBP proteins of 230 and 170 kDa (LTBP-1) or 230 kDa (LTBP-2).
The results show that Kupffer cells partly synthesize and release TGF-beta as large latent complexes. This requires the extracellular activation of TGF-beta as a prerequisite for receptor binding and cellular signaling.
背景/目的：库普弗细胞（肝脏驻留巨噬细胞）通过合成和分泌转化生长因子-β（TGF-β）对肝脏疾病的持续发展起重要作用。在某些细胞类型中，TGF-β以一种大的潜伏复合物形式表达，除了N端TGF-β前体（潜伏相关肽）外，还包含潜伏TGF-β结合蛋白（LTBP）。本研究旨在鉴定大鼠库普弗细胞中LTBP的表达情况。
通过胶原酶-链霉蛋白酶再灌注从大鼠肝脏中分离细胞，纯化并在标准条件下培养。使用碱性磷酸酶-抗碱性磷酸酶免疫染色、逆转录-聚合酶链反应以及对代谢标记蛋白进行免疫沉淀来表征TGF-β和LTBP的表达。
用抗LTBP-1（ab 39）和LTBP-2抗血清对库普弗细胞进行免疫染色表明，除了潜伏相关肽和TGF-β的表达外，两种LTBP异构体均有表达。通过逆转录-聚合酶链反应可检测到三种LTBP异构体（LTBP-1、-2、-3）和TGF-β异构体（TGF-β-1、-2、-3）的转录本。最近在肝星状细胞中描述过的缺失部分蛋白酶敏感铰链区的LTBP-1D剪接变体在库普弗细胞中也有表达。用[35S]-甲硫氨酸/半胱氨酸对库普弗细胞进行代谢标记，然后使用抗LTBP-1和LTBP-2抗血清对条件培养基进行免疫沉淀，结果表明分泌出了含有230 kDa和170 kDa（LTBP-1）或230 kDa（LTBP-2）的LTBP蛋白的高分子量TGF-β复合物。
结果表明，库普弗细胞部分合成并释放大的潜伏复合物形式的TGF-β。这需要TGF-β的细胞外激活作为受体结合和细胞信号传导的前提条件。
