Matrix metalloproteinase MMP-27 is retained in the endoplasmic reticulum by its C-terminal extension and is expressed by macrophages in the human endometrium and in endometriotic lesions
(2014)
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- Matrix metalloproteinases (MMP) are enzymes that are collectively able to cleave all the peptides within the extracellular matrix (ECM) as well as an increasing number of non-matrix proteins such as growth factors, cytokines or even intracellular proteins. They play key roles in several processes including morphogenesis, wound healing and menstrual shedding. They regulate cell proliferation, survival and migration, angiogenesis and bioactivity of other proteases. Abnormal MMP expression can lead to cancer development, rheumatoid arthritis or endometriosis. By comparison with other MMPs which are documented in thousands of research articles, MMP-27 is a new family member extremely poorly characterized. Our previous identification of MMP-27 as a gene expressed preferentially in areas of stromal breakdown in the human endometrium at menstruation prompted us to question whether this family member had specific features, different from those of the other MMPs expressed in the menstrual endometrium. Our two major aims were therefore to improve the general biochemical knowledge on MMP-27 and to specifically investigate the expression of MMP-27 in the human endometrium and in related diseases. We had noticed that MMP-27 produced by various cell lines was not secreted. Sequence comparison with other MMPs suggested that MMP-27 in mammals (but neither in birds nor reptiles) is prolonged by a unique C-terminal extension (CTE) partially hydrophobic but shorter than the transmembrane domain of membrane-type-MMPs (MT-MMPs). We therefore investigated the effects of the CTE on MMP-27 intracellular retention. Subcellular fractionation and/or confocal microscopy highlighted retention of endogenous MMP-27 and of tagged recombinant rMMP-27 in the endoplasmic reticulum (ER). In striking contrast, a truncated form of rMMP-27 without CTE was also localized in downstream structures along the secretory pathway (ERGIC and Golgi) and was secreted. Moreover, addition of the MMP-27 CTE to rMMP-10 (a classical secreted MMP) blocked its secretion and resulted in a subcellular localisation similar to that of rMMP-27. We further demonstrated that MMP-27 is not a transmembrane protein. Indeed, a target peptide added to the C-terminal end of rMMP-27 was not accessible to phosphorylation by cytosolic protein kinase A, and MMP-27 was not detected after surface biotinylation. MMP-27 is rather a peripheral membrane protein since endogenous or recombinant MMP-27 was found exclusively in the aqueous phase after Triton X-114 extraction. Interestingly, many MMP-27 isoforms, arising from alternative splicing or alternative start codons were identified. MMP-27 glycosylation was also approached by the identification of 3 potential N-glycosylation sites in MMP-27 sequence. In endometrial samples collected throughout the menstrual cycle, the levels of MMP-27 mRNA, measured by quantitative PCR, steadily increased during the secretory phase to culminate at the menstrual phase and decreased during the proliferative phase. MMP-27 mRNA levels seemed to be down-regulated in cancer. Furthermore, MMP-27 mRNA was also detected by in-situ hybridization, during mouse development, in isolated cells from various organs, suggesting a particular cellular origin. In agreement, MMP-27 was immunostained in the human endometrium in large cells scattered throughout the stroma and around blood vessels. These cells also expressed CD163 and CD206, two specific markers of alternatively activated macrophages. In addition, MMP-27 was also abundant in superficial endometriotic lesions (ovary, peritoneum) but not in deep endometriosis lesion (recto-vaginal wall). In parallel, we have also collected preliminary data on gene regulation, alternative splicing, proenzyme activation, and activity towards gelatin and casein. In conclusion, MMP-27 is an unusual protease retained in the endoplasmic reticulum by its specific C-terminal extension, without stable membrane anchorage. Production of MMP-27 by M2 macrophages culminates in the stroma of menstrual endometrium and in endometriotic lesions, but its role is totally unknown. Altogether, the unique features of MMP-27, including ER retention, restriction in cell origin, or generation of alternatively spliced isoforms most likely devoid of catalytic capacity, suggests that new, specific functions are expectable for MMP-27. In the absence of clear phenotype in knockout mice, the in vitro identification of potential substrates represents the next step towards a better understanding of this protease.
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Cominelli, A. (2014). Matrix metalloproteinase MMP-27 is retained in the endoplasmic reticulum by its C-terminal extension and is expressed by macrophages in the human endometrium and in endometriotic lesions. https://hdl.handle.net/2078.5/200921