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The role of DOT1L in systemic sclerosis

Systemic sclerosis is a devastating connective tissue disease with high morbidity and mortality. The pathophysiology is marked by autoimmunity, vascular changes and fibrosis of skin and internal organs. The complex interplay between various pathways in the pathogenesis is still incompletely understood. Two involved mechanisms are the activation of the canonical Wingless-type like (Wnt) signaling, and an imbalance in oxidative state leading to increased oxidative stress. Furthermore, the pathogenic role and therapeutic potential of epigenetics, such as post-translational modification of histones, is increasingly explored.

In this thesis, we search for novel modulators of fibrosis: we investigate two molecules reported to be connected to the Wnt signaling and oxidative stress: disruptor of telomerase 1 like (DOT1L) and acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A). Furthermore, we investigate the radiosafety of longitudinal mCT scanning protocols in preclinical animal models such as for murine lung fibrosis.

First, we explored the role of DOT1L in dermal fibroblasts, effector cells of fibrosis. DOT1L methylates histone 3 on the Lysine 79 position, thereby affecting gene expression programs. Human primary dermal fibroblasts were isolated from abdominal skin, and cultured with a specific inhibitor of DOT1L, EPZ-5676.  We confirmed interaction between DOT1L and Wnt signaling, as DOT1L inhibition upregulated Wnt target genes. Furthermore, we performed RNA sequencing on the dermal fibroblasts with and without DOT1L inhibition, revealing a dataset of more up- than downregulated differentially expressed genes after DOT1L inhibition, with upregulation of specific basement membrane molecules such as Col15A1 and PIK3K-signaling, a pathway already shown to be involved in fibrosis, as one of the enriched pathways.

In an in vivo model of subcutaneous bleomycin-induced fibrosis, no difference was found in skin fibrosis induction in mice with a fibroblast specific deletion of DOT1L. However, the efficacy of the deletion could not be confirmed in this model.

Secondly, we explored the role of ANP32A in fibrosis in vivo, by inducing skin inflammation and fibrosis in ANP32A-deficient mice. ANP32A, a small protein with a role in diverse physiological functions, is reported to protect against oxidative stress in other tissues by transcriptional control of ataxia-telangiectasia mutated (ATM) and could therefore have a role in fibrotic diseases like systemic sclerosis with an imbalance of oxidative stress. Moreover, ANP32A has been linked with the Wnt signaling.

ANP32A-deficient mice proved to be safeguarded against subcutaneous bleomycin-induced lung fibrosis and against weight loss during the experiment. There was an equal induction of skin fibrosis in these mice. ANP32A-deficient mice were not protected against skin and systemic inflammation induced by topical administration of imiquimod, a TLR7 and TLR8 agonist.

ANP32A loss was not associated with decreased ATM expression or increased oxidative stress in skin, in contrast to previous observations in other tissues such as cartilage and bone. The degradation enzyme bleomycin hydrolase was not altered in ANP32A-deficient lungs. The dermis of male ANP32A-deficient mice was less thick and contained less collagen, while the upper dermis of ANP32A-deficient mice contained more cells. In conclusion, ANP32A deficiency protects against subcutaneous bleomycin-induced lung fibrosis and weight loss, for which the underlying mechanisms further need to be investigated.

Lastly, we developed radiosafe protocols for longitudinal in vivo follow-up of murine models, such as bleomycin-induced lung fibrosis, with micro-computed tomography (mCT). Consecutive scanning of mice has the great advantage of precise quantification of lung involvement and follow-up of therapy effects, while less animals are needed per experiment. Since questions arose about the influence of repeated radiation on the disease models itself, we measured the effects of radiation on disease outcome and on the very radiosensitive blood cells of longitudinally scanned mice in different disease models and healthy mice. An adapted mCT protocol was developed to safely longitudinally scan murine models.

In conclusion, in this thesis, (1) the role of DOT1L in dermal fibroblasts was investigated, linking DOT1L inhibition to PI3K-AKT signaling and alterations in specific basal membrane molecules; (2) it was uncovered that loss of ANP32A protected against subcutaneous bleomycin-induced murine lung fibrosis, an important starting point for further research in fibrosis and lastly (3) lung imaging of fibrosis in murine models was supported by the development of radiosafe mCT scanning protocols

Date:1 Oct 2015 →  23 Nov 2020
Keywords:systemic sclerosis, DOT1L, fibrosis, ANP32A
Disciplines:Laboratory medicine, Palliative care and end-of-life care, Regenerative medicine, Other basic sciences, Other health sciences, Nursing, Other paramedical sciences, Other translational sciences, Other medical and health sciences
Project type:PhD project