Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds.
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ABSTRACT:
SUBMITTER: Hu MS
PROVIDER: S-EPMC6080120 | biostudies-literature | 2018 Aug
REPOSITORIES: biostudies-literature
Publications
Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds.
Hu Michael S MS Hong Wan Xing WX Januszyk Michael M Walmsley Graham G GG Luan Anna A Maan Zeshaan N ZN Moshrefi Shawn S Tevlin Ruth R Wan Derrick C DC Gurtner Geoffrey C GC Longaker Michael T MT Lorenz H Peter HP
Advances in wound care 20180801 8
<b>Objective:</b> In early gestation, fetal wounds heal without fibrosis in a process resembling regeneration. Elucidating this remarkable mechanism can result in tremendous benefits to prevent scarring. Fetal mouse cutaneous wounds before embryonic day (E)18 heal without scar. Herein, we analyze expression profiles of fetal and postnatal wounds utilizing updated gene annotations and pathway analysis to further delineate between repair and regeneration. <b>Approach:</b> Dorsal wounds from time-d ...[more]
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Project description:BackgroundAdult mammalian tissues heal injury to the skin with formation of scar; this process quickly seals an injured area, however, excessive scar formation can become a source of persistent pathology, interfering with multiple vital functions. In contrast, mammalian fetal tissue can heal without scar formation. We previously sought to model scarless healing in a rabbit fetal skin wound and identified gene products differentially expressed during fetal wound healing through PCR suppression subtractive hybridization (PCR SSH). One of these transcripts, previously identified simply as clone 11, showed putative increased expression in wounded fetal skin. This study establishes its identity as Ero1L-alpha and confirms its elevated expression in healing fetal wounds.FindingsAfter obtaining further sequence by 5' rapid amplification of cloned ends (RACE) we find that clone 11 is Ero1L-alpha. We determined that clone 11, a differentially expressed transcript in fetal wound healing, comprises the 3' untranslated region (UTR) of an approximately 4 kb transcript in rabbit tissues that corresponds to Ero1L-alpha. We showed that Ero1L-alpha is expressed predominantly as two transcripts in rabbit skin, namely a 1.6 kb transcript and the 4.0 kb transcript recovered in our PCR SSH screen via its 3' UTR sequence. However, a third transcript of 2.9 kb was also detected in Northern blots and was subsequently cloned and confirmed by 3' RACE. Knockdown of the clone 11 sequence in rabbit adult fibroblasts via siRNA resulted in significantly decreased Ero1L-alpha message expression. Increased expression of clone 11 (Ero1L-alpha) in a variety of cell types during the wound healing response was also confirmed by in situ hybridization.ConclusionsEro1L-alpha is one of the previously unknown clones identified in a PCR SSH screen for genes differentially expressed in fetal wounded tissue. In situ hybridization confirms that Ero1L-alpha shows increased expression in multiple cell types after wounding of the fetal integument.
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Project description:To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling osteoclast proliferation, differentiation, and apoptosis. Disruption of miRNA-mediated regulation alters osteoclast formation and bone resorption. Prior studies profiled miRNA expression in murine osteoclast precursors treated with RANKL for 24 hours. However, a more complete miRNA signature, encompassing early, mid and late stages of osteoclastogenesis, is wanting. An Agilent microarray platform was used to analyze expression of mature miRNAs in an enriched population of murine bone marrow osteoclast precursors (depleted of B220+ and CD3+ cells) undergoing 1, 3, or 5 days of RANKL-driven differentiation. Expression of 93 miRNAs, changed by >2 fold during early, mid, and late stages of osteoclastogenesis, were identified and sorted into 7 clusters. We validated the function and expression of miR-365, miR-451, and miR-99b, which were found in distinct clusters. Inhibition of miR-365 increased osteoclast number but decreased osteoclast size, while miR-99b inhibition decreased both osteoclast number and size. In contrast, overexpression of miR-451 had no effect. Computational analyses predicted mTOR, PI3 kinase/AKT, cell-matrix interactions, actin cytoskeleton organization, focal adhesion, and axon guidance pathways to be top targets of several miRNA clusters. This suggests that many miRNA clusters differentially expressed during osteoclastogenesis converge on some key functional pathways. Overall, our study is unique in that we identified miRNAs differentially expressed during early, mid, and late osteoclastogenesis in a population of primary mouse bone marrow cells enriched for osteoclast progenitors. This novel data set contributes to our understanding of the molecular mechanisms regulating the complex process of osteoclast differentiation.
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