Skin aging, influenced by a combination of intrinsic and extrinsic factors, leads to damage capable of altering skin functions. Among extrinsic factors, ultraviolet (UV) radiation is responsible for skin photoaging. In particular, these elements lead to an accumulation of senescent cells capable of contributing to the development of age-related pathologies such as skin cancers. Indeed, senescence is accompanied by profound morphological and molecular changes within the cell. This includes a modification of its secretome, which becomes enriched with pro-inflammatory cytokines, growth factors and extracellular matrix remodeling enzymes, altering the characteristics of tissues as they age. Nevertheless, the precise mechanisms leading to the senescent phenotype induced by UVB remain largely unknown.
In this context, the main objective of this work was to identify molecular mechanisms underlying the establishment of UVB-induced senescence in normal human dermal fibroblasts (NHDFs), mechanisms that could contribute to skin aging.
In vitro, we confirmed that repeated UVB exposures induce premature senescence in NHDFs and that this state is associated with activation of the three branches of the UPR (Unfolded Protein Response) pathway responsible for maintaining homeostasis of the endoplasmic reticulum (ER), the primary secretory compartment. These observations were supported by transcriptomic analysis, revealing regulatory elements linked to major senescence pathways and ER functions in UVB-exposed NHDFs. Subsequently, we showed that the ATF6α branch plays a central role in the occurrence of biomarkers of the UVB-induced senescent phenotype. Indeed, ATF6α invalidation not only protects against UVB-induced morphological changes, but reduces the percentage of SA-βgalactosidase (SA-βgal)-positive cells, prevents persistent DNA damage, and alters the expression of major factors of the senescence-associated secretory phenotype (SASP).
As SASP exerts, among other things, a pro-tumoral action, we sought to assess whether the conditioned medium (CM) of UVB-exposed fibroblasts invalidated for ATF6α could impact the migration and invasion potential of melanoma-derived cells. However, we did not observe any ATF6α-dependent pro-migratory or pro-invasive effects.
To highlight a potential role for ATF6α in another biological process, we exploited our transcriptomic and secretomic analyses and identified a possible effect of ATF6α on the paracrine control of the skin environment. To explore this, we focused on SASP factors (cytokines and metalloproteases) regulated by ATF6α and whose impact on the tissue environment was known. Next, we treated a reconstructed human epidermis (RHE) model with MC derived from NHDFs exposed to UVB or not, and invalidated or not for ATF6α. Surprisingly, we observed that MC from UVB-exposed NHDFs increased RHE thickness and basal keratinocyte proliferation, via an ATF6α-dependent mechanism. Finally, we identified IL8 as a major paracrine factor involved in this process, since IL-8 blockade by neutralizing antibodies prevents excessive keratinocyte proliferation.
In conclusion, we report the role of ATF6α in UVB-induced senescence as well as its impact on the preservation of skin homeostasis under stress conditions notably through the regulation of the expression of SASP components. This suggests that ATF6α and its effectors could be promising targets controlling the effects of skin aging.
Abstract: Impact of the UPR pathway on the establishment of the senescent phenotype induced by UVB
Skin aging, influenced by a combination of intrinsic and extrinsic factors, can result in damage that has the potential to alter skin functions. Among extrinsic factors, ultraviolet (UV) radiation is responsible for skin photoaging. These factors notably contribute to the accumulation of senescent cells which in turn can contribute to the development of age-related pathologies, including skin cancers. Indeed, senescence is characterized by profound morphological and molecular changes within the cell. This includes a modification of its secretome, which becomes enriched in pro-inflammatory cytokines, growth factors, and matrix-remodeling enzymes, altering tissue characteristics during aging. However, the exact mechanisms driving the senescent phenotype induced by UVB remain largely unknown.
In this context, the main objective of this work was to identify the underlying molecular mechanisms responsible for the establishment of UVB-induced senescence in normal human dermal fibroblasts (NHDFs), mechanisms that may play a role in skin aging.
In vitro, we confirmed that repeated exposures to UVB induce premature senescence of NHDFs and that this state is associated with the activation of the three branches of the Unfolded Protein Response (UPR), which are responsible for maintaining endoplasmic reticulum (ER) homeostasis, the primary cellular secretion compartment. These observations were supported by transcriptomic analysis, revealing regulatory elements related to major senescence pathways and ER functions in UVB-exposed NHDFs. Subsequently, we demonstrated that the ATF6α branch plays a central role in the development of the UVB-induced senescent phenotype. Indeed, the silencing of ATF6α not only protects against morphological changes induced by UVB, but also reduces the percentage of senescence-associated β-galactosidase (SA-βgal) positive cells, prevents the persistence of DNA damage, and alters the expression of major factors associated with the senescence-associated secretory phenotype (SASP).
The SASP, exerting a pro-tumoral action, led us to assess whether the conditioned medium (CM) from UVB-exposed fibroblasts invalidated for ATF6α could impact the migration and invasion potential of melanoma cells. However, we did not observe any ATF6α-dependent pro-migratory or pro-invasive effects.
To highlight a potential role of ATF6α in another biological process, we further analyzed our transcriptomic and secretomic analyses and identified a possible effect of ATF6α on the paracrine control of the skin environment. To explore this, we focused on SASP factors (cytokines and metalloproteinases) regulated by ATF6α and whose impact on tissue environment was known. Subsequently, we treated a reconstructed human epidermis (RHE) model with CM from NHDFs exposed or not to UVB and invalidated or not for ATF6α. Surprisingly, we observed that the CM from UVB-exposed NHDFs increased the thickness of the RHE as well as the proliferation of basal keratinocytes, via an ATF6α-dependent mechanism. Finally, we identified IL8 as a major paracrine factor involved in this process, as blocking IL-8 with neutralizing antibodies prevented excessive proliferation of keratinocytes. In conclusion, we report the role of ATF6α in UVB-induced senescence and its impact on the preservation of skin homeostasis under stress conditions, particularly through the regulation of the expression of SASP components. This suggests that ATF6α and its effectors could be promising targets for controlling the effects of skin aging.
