Glucocorticoid Treatment Leads to Aberrant Ion and Macromolecular Transport in Regenerating Zebrafish Fins

Abstract

Long-term glucocorticoid administration in patients undergoing immunosuppressive and anti-inflammatory treatment is accompanied by impaired bone formation and increased fracture risk. Furthermore, glucocorticoid treatment can lead to impaired wound healing and altered cell metabolism. Recently, we showed that exposure of zebrafish to the glucocorticoid prednisolone during fin regeneration impacts negatively on the length, bone formation, and osteoblast function of the regenerate. The underlying cellular and molecular mechanisms of impairment, however, remain incompletely understood. In order to further elucidate the anti-regenerative effects of continued glucocorticoid exposure on fin tissues, we performed proteome profiling of fin regenerates undergoing prednisolone treatment, in addition to profiling of homeostatic fin tissue and fins undergoing undisturbed regeneration. By using LC-MS (liquid chromatography-mass spectrometry) we identified more than 6,000 proteins across all tissue samples. In agreement with previous reports, fin amputation induces changes in chromatin structure and extracellular matrix (ECM) composition within the tissue. Notably, prednisolone treatment leads to impaired expression of selected ECM components in the fin regenerate. Moreover, the function of ion transporting ATPases and other proteins involved in macromolecule and vesicular transport mechanisms of the cell appears to be altered by prednisolone treatment. In particular, acidification of membrane-enclosed organelles such as lysosomes is inhibited. Taken together, our data indicate that continued synthetic glucocorticoid exposure in zebrafish deteriorates cellular trafficking processes in the regenerating fin, which interferes with appropriate tissue restoration upon injury.

The transcriptome of regenerating zebrafish scales identifies genes involved in human bone disease

“…We observed a skew towards upregulation of genes (n = 514 compared to n = 90 downregulated); consistent with regenerating tissues such as the caudal fin (Padhi, Joly et al, 2004, Schmidt, Geurtzen et al, 2019) (figure 1D). The osteoblast transcription factor sp7(osxterix) was substantially upregulated (2.39 log2 fold change) in regenerating scales (figure 1D), but 128 genes showed a larger up regulation than sp7.…”

Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations

“…Prednisolone (PD) is widely reported to induce glucocorticoid induced osteoporosis (GIOP) in many species and to diminish osteoblast activation following loading [ 41 ]. In zebrafish specifically, larval treatment with PD leads to reduced mineralisation [ 25 ], while in adults, treatment leads to impaired skeletal regeneration [ 42 , 43 ]. Retinoid acid (RA) plays a complex role in the skeleton, as a key factor in morphogenesis it is controlling precursor cell fate by forming a tightly controlled gradient.…”

“…These differences need to be considered when especially investigating potential drug targets that focus on trabeculation and bone marrow integrity rather than osteoanabolic cell response. When focussing on amending osteoblast function to discover putative osteoactive biomolecules that could act on validated HBM genes, assays could be performed in larvae [ 60 ], on ex vivo cultured elasmoid scales [ 28 , 29 ], or administered to adult fish to test effects on the skeleton during regeneration/bone healing in exoskeletal elements [ 43 ].…”

Non-Genomic AhR-Signaling Modulates the Immune Response in Endotoxin-Activated Macrophages After Activation by the Environmental Stressor BaP

“…The protein concentrations of the samples were determined utilizing the Pierce 660 nm proteins assay (Thermo Fisher, USA). For global proteome analysis, 30 µg protein were pre-fractionated by SDS-PAGE as previously described ( 28 ). Each sample lane was cut in 5 slices with approximately the same protein amount after Coomassie staining.…”

“…For proteome analysis, a previously described bipartite linear 55 min gradient starting from 4 % eluent B (0.1 % FA in 80 % ACN) in eluent A (0.1 % FA in water) to 55 % eluent B via 30 % eluent B after 47.5 min was used ( 28 ). After each sample, the column was flushed to 99% eluent B and reconstituted to starting conditions.…”