Ben-Eltriki Mohamed, Ahmadi Amir Reza, Nakao Yuya, Golla Kalyan, Lakschevitz Flavia, Häkkinen Lari, Granville David J, Kim Hugh
Centre for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.
Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
J Periodontal Res. 2024 Feb;59(1):94-103. doi: 10.1111/jre.13190. Epub 2023 Oct 24.
To gain insights into how proteases signal to connective tissues cells in the periodontium.
The connective tissue degradation observed in periodontitis is largely due to matrix metalloproteinase (MMP) release by gingival fibroblasts. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined.
Human gingival crevicular fluid (GCF) samples were obtained from sites with periodontal disease and healthy control sites. GzmB was quantified in the GCF ([GzmB] ) by ELISA. Gingival fibroblasts (GF) were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by ELISA to quantify GzmB-induced release of interstitial collagenase (MMP-1). In some experiments, cells were pre-treated with the inhibitor PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Systemic MMP-1 levels were measured in plasma from wild-type (WT) and granzyme-B-knockout (GzmB ) mice.
The [GzmB] in human samples was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and localized matrix degradation. GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2, which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 secretion was also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the increase in MMP-1 secretion by GF. Circulating MMP-1 was similar in WT and GzmB mice, suggesting that GzmB's effects on MMP-1 release are not reflected systemically.
These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 secretion is upregulated in a PAR1- and Erk1/2-dependent manner.
深入了解蛋白酶如何向牙周结缔组织细胞发出信号。
牙周炎中观察到的结缔组织降解很大程度上归因于牙龈成纤维细胞释放基质金属蛋白酶（MMP）。颗粒酶B（GzmB）是一种丝氨酸蛋白酶，其在牙周炎中的作用尚不清楚。
从患有牙周疾病的部位和健康对照部位获取人牙龈沟液（GCF）样本。通过酶联免疫吸附测定（ELISA）对GCF中的GzmB进行定量（[GzmB]）。在存在或不存在重组GzmB的情况下培养牙龈成纤维细胞（GF）。通过ELISA分析培养上清液，以定量GzmB诱导的间质胶原酶（MMP-1）释放。在一些实验中，细胞用抑制剂PD98059预处理以阻断MEK/ERK信号传导。在GzmB刺激之前，用ATAP-2中和抗体阻断蛋白酶激活受体-1（PAR-1）。在野生型（WT）和颗粒酶B基因敲除（GzmB-/-）小鼠的血浆中测量全身MMP-1水平。
与健康对照部位相比，人样本中牙周疾病（牙龈炎/牙周炎）部位的[GzmB]高约4-5倍，表明GzmB与局部基质降解之间存在关联。GzmB诱导培养的成纤维细胞中MMP-1分泌增加约4-5倍。GzmB诱导Erk1/2磷酸化，这被PD98059消除。PD98059也降低了GzmB诱导的MMP-1分泌上调。ATAP-2对PAR-1功能的阻断消除了GF中MMP-1分泌的增加。WT和GzmB-/-小鼠循环中的MMP-1相似，表明GzmB对MMP-1释放的影响未在全身反映出来。
这些数据表明成纤维细胞中存在一种新的由GzmB驱动的信号通路，其中MMP-1分泌以PAR1和Erk1/2依赖性方式上调。
