CD9 regulates keratinocyte migration by negatively modulating the sheddase activity of ADAM17

Abstract

CD9 is a trans-membrane protein, and has recently been implicated in different physiological and cellular processes, such as cell migration and adhesion. According to previous study, down-regulation of CD9 contributes to keratinocyte migration, critical for wound re-epithelialization. Nevertheless, it is widely believed that tetraspanin CD9 does not have ligands or function as the cell surface receptor, rather it is thought to associate with other transmembrane molecules, thereby mediate keratinocyte migration. Little is known about how CD9 associates with transmembrane molecules in migratory keratinocytes. Here, using confocal microscopy, we observed that tetraspanin CD9 and ADAM17 co-localized on the surface of keratinocytes in the course of wound repair in vivo and in vitro. Co-immunoprecipitation experiments demonstrated a direct association between CD9 and ADAM17 in HaCaT cells and C57-MKs. Functional studies revealed that down-regulation or over-expression of CD9 exerted negative regulatory effects on ADAM17 sheddase activity. This activity is involved in CD9-regulated cell motility and migration. Further studies found that ADAM17 inhibitor-TAPI-2 or siADAM17 significantly abolished the enhanced effect of keratinocyte migration induced by CD9 down-regulation. Meanwhile, the sheddase activity of ADAM17 was inhibited by TAPI-2, which decreased this release of AREG and HB-EGF in CD9-silenced HaCat cells and C57-MKs. Importantly, neutralizing antibody against HB-EGF significant weakened keratinocyte migration and motility in CD9-silenced keratinocytes, and the inhibition of CD9-regulated keratinocyte migration by siADAM17 was rescued by addition of recombinant HB-EGF, activating EGFR/ERK pathway. Collectively, our results suggest that ADAM17 sheddase activity is activated by down-regulation of CD9, thereby mediating shedding of HB-EGF and activation of EGFR/ERK signaling, which crucially affects the keratinocyte migration and wound healing.

Tetraspanin CD9 affects HPV16 infection by modulating ADAM17 activity and the ERK signalling pathway

“…5a). In agreement with earlier studies [31,33], our immunofluorescence analyses demonstrated microscopic overlap between CD9 and ADAM17.…”

“…As a member of the tetraspanin family, CD9 closely interacts with numerous membrane proteins. In particular, the direct interaction between CD9 and the transmembrane metalloproteinase ADAM17 has been reported in different cell types, including leukocytes, endothelial cells and keratinocytes [31][32][33]48]. Through this direct association, CD9 has been shown to exert negative regulatory effects on the sheddase activity of ADAM17, which is responsible for ectodomain shedding from a large variety of substrate transmembrane proteins, including adhesion molecules, cytokines, chemokines, growth factor receptors, and multiple ERK-activating growth factors, including TGFα, HB-EGF, epiregulin and amphiregulin [30,[53][54][55].…”

“…As CD9 influences ADAM17-activity [31- 33,48] as well as ERK signalling [49,50] (see also Kummer et al, in the same issue [62]), and ADAM17 regulates HPV16 infection via the ERK1/2 signalling pathway [12], we expected that the cellular CD9 level likewise would influence ERK1/2 activation in our cell model. Indeed, modulation of the endogenous CD9 level in HeLa cells resembles the results obtained in HPV16 PsVs infection assays (for infection assays in CD9 overexpressing HeLas see Figs.…”

“…Furthermore, it has been well established that CD9 also regulates the function of cell membrane proteases including ADAM17 [28,30,31,33]. ADAM17, in turn, triggers ERK signalling which is important for HPV16 entry receptor formation [12].…”

“…In this regard, Tsukamoto et al reported that CD9 negatively regulates the shedding of the substrate LR11, a member of the low-density lipoprotein receptor family which has a key role in cell migration, adhesion, and drug resistance, in various leukaemia cell lines [32]. Furthermore, Liu et al have recently shown the direct association of CD9 with ADAM17 in keratinocytes and confirmed that CD9 exerts negative regulatory effects on this metalloproteinase resulting in diminished shedding of its substrate heparin-binding epidermal growth factor (HB-EGF) and reduced activation of EGFR/ERK signalling pathway, crucially affecting keratinocyte migration and wound healing [33].…”