Potential role of ANKRD1 in senescence mediated wound healing

我们已经通过RNA-seq和全细胞质谱分析证明ANKRD1的表达随着衰老而上升，这表明ANKRD1在衰老的WI-38人二倍体成纤维细胞中高度表达。此外，通过转染小干扰（si） rna，在衰老前成纤维细胞中沉默ANKRD1，证实沉默ANKRD1可以减少衰老。我们发现，ANKRD1的缺失降低了衰老标志物的水平，包括p16和p21 mrna和SA-半乳糖苷酶。这些数据表明，沉默ANKRD1可以延缓衰老。我们也开始在人体皮肤样本中检测到ANKRD1。人体皮肤样品由Dr. C. Chia （LCI）提供，用ANKRD1 （LGG）进行初始染色。抗ankrd1抗体初步IF染色显示，零星分布的真皮细胞有较强的核染色；较弱的信号广泛存在于表皮，从基底层到角质层。

We have documented that ANKRD1 expression rises with senescence by RNA-seq and whole-cell mass spectrometry analyses, which have revealed that ANKRD1 is being highly expressed in senescent WI-38 human diploid fibroblasts. Moreover, silencing ANKRD1 reduces senescence, as determined by silencing ANKRD1 in pre-senescent fibroblasts by transfecting small interfering (si)RNAs. We found that depletion of ANKRD1 decreased the levels of senescent markers including p16 and p21 mRNAs and SA--galactosidase. These data indicate that silencing ANKRD1 attenuates senescence. We have also began to detect ANKRD1 in human skin samples. Human skin samples were provided by Dr. C. Chia (LCI) for initial staining with ANKRD1 (LGG). Preliminary IF staining with anti-ANKRD1 antibody showed a strong nuclear staining in sporadically localized dermal cells; weaker signals were observed broadly in the epidermis, from the basal layer to the lower stratum corneum. Ongoing Efforts: Towards Objective 1, we are investigating the mechanisms of ANKRD1 expression in senescence and ANKRD1 impact on the SASP trait. We expect to find either elevated transcription or increased ANKRD1 mRNA stability in senescent cells, as well as RNA-binding proteins, miRNAs, or both involved in the post-transcriptional regulation of ANKRD1 expression (LGG). We are also investigating how ANKRD1 affects the production and secretion of SASP factors, particularly SASP factor platelet-derived growth factor AA (PDGF-AA), which is required for wound healing. We expect that ANKRD1 silencing will reduce the secretion of PDGF-AA (LGG). The impact of ANKRD1 silencing or overexpression on the senescent phenotype will be investigated by RNA-seq analysis, cell proliferation, and cell survival assays. Towards Objective 2, we are assessing the levels of ANKRD1 as a function of age in mouse skin (SLAM study). Besides measuring ANKRD1 mRNA and protein levels in skin during aging, we will test the patterns of ANKRD1 expression and senescence in mouse skin during wound healing (LGG, TGB). These tissues will be used to detect senescent, ANKRD1-positive cells by IHC staining in the region of the healing wound (LGG, TGB). Towards Objective 3, we are studying the effect of ANKRD1 deficiency on the extracellular media metabolites (amino acids, biogenic amines and lipids), important in wound healing. Extracellular media metabolites (amino acids, biogenic amines and lipids) will be measured using liquid chromatography tandem mass spectrometry (LC-MS/MS). Metabolites will be extracted and concentrations are measured using The AbsoluteIDQ p500 kit (Biocrates Life Sciences AG, Innsbruck, Austria) following manufacturers protocol for a 5500 QTrap (Sciex, Framingham, MA) mass spectrometer (LGG, LCI).