Relevanz der humanen Xylosyltransferase-I in der Seneszenz-assoziierten Gewebe-Remodellierung und der adulten Knochenhomöostase

Human xylosyltransferases-I and -II (XT-I and II), which are encoded by the genes XYLT1 and XYLT2, are significantly involved in extracellular matrix (ECM) remodeling by catalyzing the initial step of proteoglycan (PG) biosynthesis. Aberrant XT-I gene expression and activity is associated with age-associated diseases such as fibrosis or osteoarthritis, but also with the occurrence of skeletal dysplasia. This study therefore investigated the relevance of XT-I in cellular senescence, an important feature of the ageing process and in the adult bone homeostasis. In the first part of this dissertation, acute senescence was successfully induced in NHDF-derived proto-myofibroblasts using H2O2 and a suppression of XT-I expression and activity together with reduced ECM expression was shown. The simultaneously detected increase in mRNA expression of the transcription factor KLF4 and the cytokine IL-1β indicates a XT-I reduction at the transcription level. These data demonstrate a significant involvement of XT-I in the development of an antifibrotic phenotype during physiological dermal wound healing. In contrast, H2O2-mediated induction of acute senescence in human cardiac stem cells (hCSCs) caused neither XT-I regulation. However, a senescence-associated secretory phenotype (SASP)-mediated induction of XT-I gene expression and activity along with an expression increase of ECM-associated proteins was determined for the first time after hCSCs were cultured with conditioned media of transforming growth factor-β1 (TGF-β1)- or TGF-β1+H2O2-treated hCSCs. Accordingly, XT-I regulation in hCSCs only occurs in TGF-β1-associated acute senescence processes, which could have an impact on the development of cardiac fibrosis. In contrast, replicative stress induced chronic senescence in hCSCs resulted in suppressed XT-I activity, reduced ECM expression and the formation of an immunosupressive SASP. The characterization of XYLT1-deficient hCSCs confirmed that XT-I suppression in chronic senescent hCSCs was directly involved in the detected effects, promoting the development of age-associated cardiac pathologies. A significantly elevated concentration of reactive oxygen species represents a potential cause for the detected effects after induction of TGF-β1-associated and chronic senescence in hCSCs. In the second part of this dissertation, based on previous studies that identified human pathogenic XYLT1 mutations as causative for the manifestation of skeletal dysplasia, inter alia Debuquois dysplasia (DBQD) type 2, the relevance of XT-I in human adult bone homeostasis was analyzed. For the first time, a XYLT1 deficiency was generated in human mesenchymal stem cells (hMSCs) using CRISPR/Cas9 gene editing. After chondrogenic differentiation of hMSCs, XYLT1 deficiency led to the assembly of an aberrant cartilage matrix, which was partly caused by reduced mRNA expression of the chondrogenic transcription factor SOX9. In addition, increased gene expression of the osteogenic transcription factor RUNX2 indicated premature ossification. After osteogenic differentiation of XYLT1-deficient hMSCs, a decrease in matrix mineralization was demonstrated by reduced calcification and extracellular phosphate concentration. Since the XYLT1-deficient osteoblasts showed reduced mRNA expression of the osteogenic markers osteopontin and osteocalcin, their involvement is conceivable. The relevance of XT-I in bone resorption was investigated using siRNA-mediated XYLT1 knockdown in human osteoclasts. A reduced bone resorption rate of the XYLT1-deficient osteoclasts was demonstrated, in which, inter alia, a reduced extracellular acid phosphatase activity was involved. A potential cause for the impaired cell functionality is a premature induction of osteoclastogenesis, indicated by a detected increase in mRNA expression of various osteoclast markers, inter alia, the osteoclast-associated receptor OSCAR and the matrix metalloproteinase MMP-9. Based on the results of this study, the clinical manifestations of DBQD type 2 patients at the human cellular level have been demonstrated for the first time. Furthermore, the data show that XT-I is highly relevant not only in embryonic skeletal development, but also in adult bone homeostasis. In summary, the findings of this work contribute to a better understanding of XT-I regulation in senescence-associated ECM remodeling and adult bone homeostasis and serve as a basis for the development of future therapeutic options for patients with pathological cardiac tissue remodeling or skeletal dysplasia.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Bielefeld

Year

2024

Author dc:creator

• Schmidt, Vanessa

Repository record source_url

https://pub.uni-bielefeld.de/record/2989960

OAI identifier oai:identifier

oai:pub.uni-bielefeld.de:2989960