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Superoxide, a form of reactive oxygen species (ROS), is catabolized by superoxide dismutase (SOD) and contributes to carcinogenesis via the oxidative damage it inflicts on cells. The aim of this research was to analyze the potential vitamin D-mediated regulation of the antioxidative “SOD1-to-SOD2 switch” within the human MG-63 osteosarcoma model. For this study, real-time PCR analysis was performed using MG-63 cells exposed to metabolically active 1,25(OH)2D3. First, a sustained statistically significant >2-fold suppression of proliferating cell nuclear antigen (PCNA) transcripts was observed after 10 nM but not at 100 nM of 1,25(OH)2D3 treatment, suggesting a cytostatic effect. In order to assess regulators of mitochondrial oxidative phosphorylation, gene expression of COX2 and COX4l1 of the mitochondrial complex IV and antioxidative enzymes (SOD1, SOD2 and Catalase (CAT)) were monitored. For COX2 and COX4l1, no changes in gene expression were observed. However, a concomitant decrease in CAT and SOD1 mRNA, and increase in SOD2 mRNA after 24 h of 10 nM 1,25(OH)2D3 treatment were observed. A ~8-fold increase in SOD2 mRNA was apparent after 48 ours. The significant increase in SOD2 activity in the presence of vitamin D indicates an antioxidant potential and sensitization of vitamin D during osteosarcoma transformation and mitochondrial detoxification over time.

The relationship between the active form of vitamin D3 (1,25‐dihydroxyvitamin D, 1,25(OH)2D) and reactive oxygen species (ROS), two integral signaling molecules of the cell, is poorly understood. This is striking, given that both factors are involved in cancer cell regulation and metabolism. Mitochondria (mt) dysfunction is one of the main drivers of cancer, producing more mitochondria, higher cellular energy, and ROS that can enhance oxidative stress and stress tolerance responses. To study the effects of 1,25(OH)2D on metabolic and mt dysfunction, we used the vitamin D receptor (VDR)‐sensitive MG‐63 osteosarcoma cell model. Using biochemical approaches, 1,25(OH)2D decreased mt ROS levels, membrane potential (ΔΨmt), biogenesis, and translation, while enforcing endoplasmic reticulum/mitohormetic stress adaptive responses. Using a mitochondria‐focused transcriptomic approach, gene set enrichment and pathway analyses show that 1,25(OH)2D lowered mt fusion/fission and oxidative phosphorylation (OXPHOS). By contrast, mitophagy, ROS defense, and epigenetic gene regulation were enhanced after 1,25(OH)2D treatment, as well as key metabolic enzymes that regulate fluxes of substrates for cellular architecture and a shift toward non‐oxidative energy metabolism. ATACseq revealed putative oxi‐sensitive and tumor‐suppressing transcription factors that may regulate important mt functional genes such as the mTORC1 inhibitor, DDIT4/REDD1. DDIT4/REDD1 was predominantly localized to the outer mt membrane in untreated MG‐63 cells yet sequestered in the cytoplasm after 1,25(OH)2D and rotenone treatments, suggesting a level of control by membrane depolarization to facilitate its cytoplasmic mTORC1 inhibitory function. The results show that 1,25(OH)2D activates distinct adaptive metabolic responses involving mitochondria to regain redox balance and control the growth of osteosarcoma cells. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

MicroRNAs (miRNAs) are a major class of conserved non-coding RNAs that have a wide range of functions during development and disease. Biogenesis of canonical miRNAs depend on the cytoplasmic processing of pre-miRNAs to mature miRNAs by the Dicer endoribonuclease. Once mature miRNAs are generated, the miRNA-induced silencing complex (miRISC), or miRISC, incorporates one strand of miRNAs as a template for recognizing complementary target messenger RNAs (mRNAs) to dictate post-transcriptional gene expression. Besides regulating miRNA biogenesis, Dicer is also part of miRISC to assist in activation of the complex. Dicer associates with other regulatory miRISC co-factors such as trans- activation responsive RNA-binding protein 2 (Tarbp2) to regulate miRNA-based RNA interference. Although the functional role of miRNAs within epidermal keratinocytes has been extensively studied within embryonic mouse skin, its contribution to the normal function of hair follicle bulge stem cells (BSCs) during post-natal hair follicle development is unclear. With this question in mind, we sought to ascertain whether Dicer-Tarpb2 plays a functional role within BSCs during induced anagen development by utilizing conditional knockout mouse models. Our findings suggest that Dicer, but not Tarbp2, functions within BSCs to regulate induced anagen (growth phase) development of post-natal hair follicles. These findings strengthen our understanding of miRNA-dependency within hair follicle cells during induced anagen development.

Osteogenesis imperfecta is an inherited disorder characterized by increased bone fragility, fractures, and osteoporosis, and most cases are caused by mutations affecting the type I collagen genes. Here, we describe a new mouse model for Osteogenesis imperfecta termed Aga2 (abnormal gait 2) that was isolated from the Munich N-ethyl-N-nitrosourea mutagenesis program and exhibited phenotypic variability, including reduced bone mass, multiple fractures, and early lethality. The causal gene was mapped to Chromosome 11 by linkage analysis, and a C-terminal frameshift mutation was identified in the Col1a1 (procollagen type I, alpha 1) gene as the cause of the disorder. Aga2 heterozygous animals had markedly increased bone turnover and a disrupted native collagen network. Further studies showed that abnormal proalpha1(I) chains accumulated intracellularly in Aga2/+ dermal fibroblasts and were poorly secreted extracellularly. This was associated with the induction of an endoplasmic reticulum stress-specific unfolded protein response involving upregulation of BiP, Hsp47, and Gadd153 with caspases-12 and -3 activation and apoptosis of osteoblasts both in vitro and in vivo. These studies resulted in the identification of a new model for Osteogenesis imperfecta, and identified a role for intracellular modulation of the endoplasmic reticulum stress-associated unfolded protein response machinery toward osteoblast apoptosis during the pathogenesis of disease.

There are numerous ways of portraying cancer complexity based on combining multiple types of data. A common approach involves developing signatures from gene expression profiles to highlight a few key reproducible features that provide insight into cancer risk, progression, or recurrence. Normally, a selection of such features is made through relevance or significance, given a reference context. In the case of highly metastatic cancers, numerous gene signatures have been published with varying levels of validation. Then, integrating the signatures could potentially lead to a more comprehensive view of the connection between cancer and its phenotypes by covering annotations not fully explored in individual studies. This broader understanding of disease phenotypes would improve the predictive accuracy of statistical models used to identify meaningful associations. We present an example of this approach by reconciling a great number of published signatures into meta-signatures relevant to Osteosarcoma (OS) metastasis. We generate a well-annotated and interpretable interactome network from integrated OS gene expression signatures and identify key nodes that regulate essential aspects of metastasis. While the connected signatures link diverse prognostic measurements for OS, the proposed approach is applicable to any type of cancer.

Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H 2 O 2 ) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H 2 O 2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H 2 O 2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H 2 O 2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H 2 O 2 –dependent pathways, EGF, FOXO1 and IKKα. This study expands on our current understanding of the clinical potential of low-level H 2 O 2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits.

Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H₂O₂) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H₂O₂ signaling. Genetic depletion of H₂O₂ in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H₂O₂ depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and upregulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.

Mice and human patients with impaired vitamin D receptor (VDR) signaling have normal developmental hair growth but display aberrant post-morphogenic hair cycle progression associated with alopecia. In addition, VDR–/– mice exhibit impaired cutaneous wound healing. We undertook experiments to determine whether the stress-inducible regulator of energy homeostasis, DNA damage-inducible transcript 4 (Ddit4), is involved in these processes. By analyzing hair cycle activation in vivo, we show that VDR−/− mice at day 14 exhibit increased Ddit4 expression within follicular stress compartments. At day 29, degenerating VDR−/− follicular keratinocytes, but not bulge stem cells, continue to exhibit an increase in Ddit4 expression. At day 47, when normal follicles and epidermis are quiescent and enriched for Ddit4, VDR−/− skin lacks Ddit4 expression. In a skin wound healing assay, the re-epithelialized epidermis in wildtype (WT) but not VDR−/− animals harbor a population of Ddit4- and Krt10-positive cells. Our study suggests that VDR regulates Ddit4 expression during epidermal homeostasis and the wound healing process, while elevated Ddit4 represents an early growth-arresting stress response within VDR−/− follicles.