Persistent or continuous exposure to UVA leads to photoaging, where the involvement of ROS is implicated. The generation of ROS is well documented on UVA irradiation [10]. We have observed that there was the generation of ROS, induction of DNA damage, depletion of antioxidant enzyme activities and lipid peroxidation in UVA exposed A375 cells; but no generation of ROS, DNA damage, lipid peroxidation, or cell killing was observed in the UVA-bystander cells (communicated). So, it was not possible to infer from those findings about the role of bystander cells in UVA-induced aging.
The integrity of the skin is largely maintained through the renewal and restructuring of damaged cells. The major factor that contributes to the injury of cells is through DNA damage from UV irradiation. Deficiency in DNA repair has been associated with skin aging. Cell cycle arrest halts the propagation of dysfunctional cells initiated by different detrimental stimuli. Blockage in the cell cycle has been linked with increased senescence from UV-mediated photoaging [11]. In fibroblast, UVA exposure led to cell cycle arrest and skin aging [12]. We have therefore observed the distribution of cells in the different phases at various times (0, 6, 12 and 24 h) after exposure to UVA-CM to assess the influence on cell cycle progression in the UVA-bystander A375 cells. The findings are shown in Fig. 1. In the UVA-bystander cells, by 6 h after the CM conditioning treatment, the G1/S cell population increased to (80.17 ± 0.29)% from (69.69 ± 0.62)% in control cells. This accumulation of cells in the G1/S phase persisted until 24 h. We have seen earlier that UVA also induced G1/S arrest in A375 cells [8]. Mao et al. had observed G1 arrest in replicative senescent fibroblasts population [13]. Although cellular senescence is different, it is also associated with another form of growth arrest, known as quiescence; here arrest occurs at G1 and possibly also in the G2 phase. Despite no induced DNA damage, the observed delay in cell cycle on exposure to UVA-CM suggests alteration of cell function that may be related to cellular photoaging in the UVA-bystander cells.
The MMPs contribute to the degradation of the extracellular matrix (ECM), significant amongst them are MMP-1, -3 and − 9 that have a direct correlation with photoaging [2]. UVA irradiation induces activation of transcription factors like AP-1 and NF-κB, which are involved in the transcriptional regulation of MMPs [14]. UVA and UVB induce upregulation of MMP-1 in different cell lines [15]. MMP-1 is a multifunctional protein that is highly related to stress induced premature senescence and photo-induced wrinkling [3]; it degrades collagen 1 in the ECM. Inhibiting the expression of MMP-1 prevented photoaging in cells [16]. We found that exposure to UVA increased the expression of the MMP-1 gene. UVA-bystander A375 cells also exhibited an increase, but it was less pronounced than in UVA exposed cells (Fig. 2a). Our findings reflect the possible involvement of UVA-bystander cells in photoaging through the induction of expression of MMP-1.
Stromelycin1 or MMP-3 is known for its role in inflammation. It also degrades collagen IV, V, IX, X, gelatin, laminin, fibronectins and also activates pro-collagenase, like pro-MMP-9 [3]. Activated MMP-9 (gelatinase-B) has a role in various physiological processes, where its function in the degradation of the ECM is important. MMP-9 can degrade elastin and hydrolyze collagen type IV in the skin [3]. Both MMP-3 and − 9 were overexpressed in UVA irradiated cells [17]. We also found upregulation of MMP-3 and MMP-9 mRNA on UVA irradiation. In the UVA-bystander cells, the expression of both these proteases was suppressed beyond that present in the control cells, as shown in Fig. 2b and 2c, respectively. The MMP-9 promoter contains a binding site for the NF-κB [14]. NF-κB was not induced in the UV-bystander A375 cells [18]. This may explain the transcriptional downregulation of MMP-9.
MMP-3 and MMP-9 are also biomarkers of cancer, due to their role in tumour growth, invasion and metastasis [19]. Inhibition of MMP-3 and MMP-9 can block metastasis, angiogenesis and cancer [20]. The downregulation of MMP-3 and − 9 indicated that these proteases do not contribute to photoaging in bystander cells and also suggested that the UVA-bystander effect does not increase the propensity towards cancer. This is in accordance with our earlier findings that there was no increase in the side population of cancer stem cells in the UVA-bystander cells (communicated).
Exposure to UVA activates inflammatory responses due to oxidative stress via intracellular signaling pathways in the skin. Biochemical, genetic and pharmacologic evidence indicates the pivotal role of COX-2 in inflammation. Hence, UVA-mediated signaling that induces the expression of COX-2 could be a target for preventing skin inflammation [21]. COX-2 gene transcription is elevated by AP-1, which binds to a specific site on the COX-2 gene [22]. COX-2 also activated NF-κB signaling pathway in UV irradiated cells [22]. It has been demonstrated that COX-2 expression is significantly upregulated in photoaged skin; it positively correlated with the degree of solar elastosis [5]. Inhibitors of COX-2 have been found to prevent photoaging [23]. A significant upregulation in the expression of COX-2 in both, UVA irradiated cells and also in the UVA-bystander cells was observed (Fig. 2d), the extent of upregulation of COX-2 was, however much lower in the bystander cells. Through the induction of COX-2, therefore, the bystander cells had a direct role in photoaging.
Collagen provides strength and elasticity to the skin. Accumulation of degraded collagen leads to the formation of wrinkles, which is a characteristic of photoaging [4]. An increase in the expression of MMP-1 results in the breakdown of the collagen fibers. The main building blocks responsible for the de novo synthesis of collagen fibers are collagen type 1 (COL1A1). UV radiation significantly downregulates collagen through the AP-1 signaling pathway [22]. The degradation of collagen and elastin and the increase of MMPs are inversely proportional [22]. An increase in expression of MMP-1 is associated with downregulation of type I collagen protein in photoaged skin [24]. Type I and III fibrillar collagens are degraded by MMP-1; proMMP- 9, on activation by MMP-3 can further degrade collagen fragments generated by MMP-1. A decrease in expression of the COL1A1 gene was evident in both UVA exposed cells and also in the bystander cells (Fig. 2e). The decrement in the expression of COL1A1 in the bystander cells was less pronounced than that in the UVA irradiated cells.
Elastin is another important component of the ECM. Its presence in connective tissues imparts elasticity to the skin. Elastin is rich in alanine, glycine, leucine, proline and valine, which are arranged in short repeated sequences to form a highly flexible and dynamic structure. Solar elastosis arises from the degradation of elastic fibers that is integral to cutaneous photoaging [25]. Degradation of elastin leads to wrinkle and line formations in the skin [25]. The expression of elastin was remarkably downregulated in both UVA exposed cells and in its bystander cells (Fig. 2f). The reduction in the expression of elastin being more marked in the irradiated cells than in the UVA-bystander cells. The findings that both COL1A1 and elastin are downregulated in the A375-bystander cells further endorsed the contribution of bystander cells to photoaging.