Macotela Yazmín, Aguilar Manuel B, Guzmán-Morales Jessica, Rivera José C, Zermeño Consuelo, López-Barrera Fernando, Nava Gabriel, Lavalle Carlos, Martínez de la Escalera Gonzalo, Clapp Carmen
Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, México.
J Cell Sci. 2006 May 1;119(Pt 9):1790-800. doi: 10.1242/jcs.02887. Epub 2006 Apr 11.
The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.
催乳素的16 kDa N端片段(16K-催乳素)是一种有效的抗血管生成因子。在此,我们证明软骨细胞产生并分泌的基质金属蛋白酶(MMPs)可从全长催乳素生成具有生物功能的16K-催乳素。在中性pH条件下与人催乳素一起孵育时,软骨细胞提取物、条件培养基以及培养中的软骨细胞会切割催乳素中的Ser155-Leu156肽键,在分子内二硫键还原后产生一个16 kDa的N端片段。这种16K-催乳素在体外可抑制碱性成纤维细胞生长因子(FGF)诱导的内皮细胞增殖。Ser155-Leu156位点高度保守,人和大鼠的催乳素均可被来自任一物种的软骨细胞在此位点切割。MMP抑制剂EDTA、GM6001或1,10-菲咯啉可完全消除软骨细胞裂解物将催乳素转化为16K-催乳素的过程。纯化的MMP-1、MMP-2、MMP-3、MMP-8、MMP-9和MMP-13在Gln157处切割人催乳素,该位点位于软骨细胞蛋白酶切割位点下游一个残基处,其相对活性如下:MMP-8 > MMP-13 > MMP-3 > MMP-1 = MMP-2 > MMP-9。最后,软骨细胞表达催乳素mRNA(通过RT-PCR显示),并且含有并释放抗血管生成的N端16 kDa催乳素(通过蛋白质印迹和内皮细胞增殖检测)。这些结果表明,软骨中的几种基质金属蛋白酶可从全身来源或局部产生的催乳素生成抗血管生成的16K-催乳素。