Pathobiochemical impact of human xylosyltransferase-I in bone homeostasis

蛋白聚糖（PG）是骨的细胞外基质（ECM）的组成部分，并且通过调节生长因子和细胞因子的生物利用度来调节骨重塑细胞的增殖以及分化。此外，PG定义胶原原纤维组装，并因此对骨骼的机械完整性产生影响。PG-糖基化的限速步骤由人木糖基转移酶-I和-II（XT-I和-II）催化。正如我们小组最近发表的，血清XT活性反映了PG合成速率，因此是一种强大的非侵入性生物标志物。在我们的第一个资助期，我们证明了XT-I缺乏与原代人皮肤成纤维细胞的异常ECM重塑和细胞衰老有关。我们由此产生的假设，一个强大的病理生理学相关性的XT-I-缺陷得到加强了几个致病性XYLT 1突变与骨骼发育不良相关的证据。因此，本研究的目的是利用原代间充质干细胞（hMSC）和破骨细胞来阐明XT-I在骨稳态中的病理生物化学影响。为了评估XT-I缺陷对hMSC的细胞功能的影响，我们将进行基于CRISPR-（成簇规则间隔短回文重复序列）-Cas9的XYLT 1敲除。基因编辑的hMSC将进一步用于研究这些细胞的分化潜力以及由hMSC产生的成骨细胞和软骨细胞的ECM重塑。在第二个项目中，我们将研究XT-I缺乏对破骨细胞功能的影响。此外，我们将分析血清中的XT活性是否有资格成为骨质疏松症和异常骨质溶解的生物标志物。在最后一期中，我们将通过研究hMSC老化过程中XT的调节以及XT-I缺陷的hMSC的表型来密切关注XT-I缺陷与细胞衰老的关系。从长远来看，我们的结果不仅有助于更好地理解骨稳态的分子机制，而且还将揭示XT相关骨骼疾病的假定治疗干预措施。

Proteoglycans (PG) are component parts of the extracellular matrix (ECM) of bone and regulate proliferation as well as differentiation of bone-remodeling cells by modulating the bioavailability of growth factors and cytokines. Furthermore, PG define collagen fibril assembly and consequently exert influence on mechanical integrity of the skeleton. The rate-limiting step of PG-glycosylation is catalyzed by human xylosyltransferase-I and -II (XT-I and -II). As recently published by our group, serum XT activity reflects the PG synthesis rate and thus is a powerful non-invasive biomarker.In the course of our first funding period, we demonstrated that XT-I-deficiency is associated with abnormal ECM remodeling and cellular senescence in primary human dermal fibroblasts. Our resulting hypothesis of a strong pathophysiological relevance of XT-I-deficiency gets reinforced by the descripton of several pathogenic XYLT1 mutations being associated with skeletal dysplasia. So the aim of this study is to unravel the pathobiochemical impact of XT-I in bone homeostasis using primary human mesenchymal stem cells (hMSC) and osteoclasts.In total, our study addresses four issues. To evaluate the influence of XT-I-deficiency on cellular functionality of hMSC, we will perform a CRISPR-(clustered regularly interspaced short palindromic repeats)-Cas9-based XYLT1 knockout. Gene edited hMSC will further be used to study the differentiation potential of these cells as well as the ECM remodeling of osteoblasts and chondrocytes generated from hMSC. In the second project we will examine the influence of XT-I-deficiency on cellular functionality of osteoclasts. In addition, we will analyze whether serum XT-activity qualifies to be a biomarker for osteoporosis and aberrant osteolysis. In the last issue we will look closely at the association of XT-I-deficiency and cellular senescence by studying XT regulation during ageing of hMSC and by characterizing the phenotype of XT-I-deficient hMSC.For the long term our results will not only contribute to a better understanding of molecular mechanisms of bone homeostasis but will also reveal putative therapeutic interventions for XT-associated skeletal diseases.