Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.

Originally simply reported to be in a stable and irreversible growth arrest in vitro , senescent cells are now clearly associated with normal and pathological ageing in vivo . They are characterized by several biomarkers and changes in gene expression that may depend on epigenetic factors, such as histone acetylation, involving a balance between histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we investigate the expression and the role of HDACs on the senescent phenotype of dermal fibroblasts. We report that during replicative senescence, most canonical HDACs are less expressed. Moreover, treatment with SAHA, a histone deacetylase inhibitor (HDACi) also known as Vorinostat, or the specific downregulation of HDAC2 or HDAC7 by siRNA, induces the appearance of senescence biomarkers of dermal fibroblasts. Conversely, the ectopic re-expression of HDAC7 by lentiviral transduction in pre-senescent dermal fibroblasts extends their proliferative lifespan. These results demonstrate that HDACs expression can modulate the senescent phenotype, highlighting their pharmaceutical interest in the context of healthy ageing.

No abstract

Collagen XVII (COL17/BP180) is a hemidesmosmal type II transmembrane protein between the epidermis and dermis which can be targeted by autoantibodies in autoimmune blistering disorders, including linear IgA bullous dermatosis (LAD). The ectodomain of COL17 is known to be physiologically or pathologically cleaved within the juxtamembranous noncollagenous NC16A region, which results in the 120kD (LAD-1) and the further C-terminally processed 97kD (LABD-97) ectodomains. Significantly, LAD autoantibodies preferentially target LAD-1 and LABD-97 but not full-length COL17, indicating proteolysis within the NC16A domain induces neoepitopes on the cleaved COL17 ectodomains. However, little is known about how neoepitopes are produced on processed COL17, especially in relation to C-terminal cleavage. This study shows that not only ectodomain shedding but also cleavage of the Cterminus is associated with development of conformational neoepitopes on proteolyzed COL17. We first produced a novel monoclonal antibody (mAb) that specifically reacts with LAD-1 and LABD-97, but not with full-length COL17. The epitope of the mAb was mapped on the 15th collagenous domain of the COL17 ectodomain, indicating comformational neoepitopes are induced in this region after ectodomain shedding. To investigate the roles of C-terminal cleavage in the development of neoepitopes, we produced a recombinant COL17 (COL17-DC; Met 1-Arg 1174) with a C-terminus 323 amino acids shorter than the full length COL17 (Met 1-Pro 1497). Interestingly, the mAb reacted with COL17-DC, suggesting cleaving only the C-terminal of COL17 also induces structural changes and exposes comformational neoepitopes on the 15th collagenous domain. Notably, the LAD autoantibodies also reacted with COL17-DC, indicating C-terminal processing of COL17 is associated with the autoimmunity of LAD. The present study shows that proteolysis of COL17 induces dynamic structural changes on proteolyzed COL17 and conformational neoepitopes, which are targeted by LAD autoantibodies.