As acutely or single treated OTCs showed no changes in histology, chronic UV-irradiation led to morphological changes in OTCs with NHEKs. Here, OTCs revealed decreased filaggrin protein levels accompanied with an increase of the wound-associated protein decorin. These findings were previously confirmed in solar-irradiated skin (Scott 1986; Yeo et al. 1991), arguing that the OTC irradiation model well represents the in vivo situation.
Different effects can be found after chronic UVA or UVA+B-irradiations in OTCs. In general, UVA and UVB-light are supposed to cause different reactions in the skin. While UVA-irradiation can penetrate deep into the dermal layer of the skin, UVB-light is known to be directly absorbed in the epidermal layer (Ichihashi et al. 2003). However, solar radiation is composed of both UVA and light. Therefore, besides UVA-radiation we additionally included the composition of UVA and
UVB-Discussion
102 light (UVA+B) into the study of the telomere length regulation in order to simulate the in vivo situation more accurately.
When characterizing the chronic UV-treated NHEK OTCs, we found that increasing doses of chronic UVA-exposure caused increased cell death of dermal fibroblasts followed by epithelial atrophy. In contrast, chronic UVA+B-irradiation resulted in less dermal cell death indicated by maintenance of a well-stratified epidermis. Importantly, this suggested that UVA+B, actually representing the in vivo situation more accurately, treatment is less harmful to OTCs.
Interestingly, Bernerd and colleagues showed a clear reduction of fibroblast number already after 24 and 48 h with a single UVA or UVA+B-treatment (Bernerd & Asselineau 1997; Bernerd & Asselineau 1998; Bernerd et al. 1999; Bernerd et al. 2003; Duval et al. 2003; Bernerd & Asselineau 2008). This is surprising as in our hands only UVA caused a clear reduction in the number of fibroblasts, while 4 week long UVA+B-treatments still showed a rather “unaltered” fibroblasts profile.
Moreover, different from our findings but by using a similar multiple UV-irradiation approach, Huang and colleagues showed in human engineered skin no morphological changes by UVA, UVB and UVA+B-irradiation (Huang et al. 2009). An important difference between this study and the present analysis, however, was the time schedule of irradiation. While the skin model by Huang et al. was UV-treated four times for one week, our OTCs were treated three times per week for a total time of four weeks, resulting in about a 3-4 times higher total UV-dose. Thus, the effect on morphology might be time-dependent and only be detectable when administering a continuous irradiation
When UV-irradiating OTCs with HaCaT cells, a different response compared to OTCs with NHEK was observed. Interestingly, chronic UVA- or UVA+B-treatments did not alter the number of fibroblasts. Because HaCaT cells are tetraploid, it might be that the HaCaT cell epidermis is absorbing more UV-irradiation than NHEK epithelia. But also a different cross-talk between fibroblasts and HaCaT cells might be possible, inducing other protein expression which might have resulted in more resistant fibroblasts upon UV-exposure.
Taken together, we found that chronic UVA+B exposure was less harmful to keratinocytes and fibroblasts than chronic UVA-irradiation. This suggests that the cellular regulation can be different depending on the UV composition. Thus, we cannot conclude from data acquired with UVA- or UVB-light only.
Telomere maintenance after chronic UV-irradiation is dependent on a functional p53 pathway in keratinocytes. Telomere shortening in keratinocytes was shown in sun-exposed skin compared to sun-protected skin sides (PhD thesis Dr. D. Krunić 2008, published online, Ikeda et al., 2014). Given that this shortening might need protracted exposure, the exact mechanism of UV-induced telomere shortening in human skin remains elusive. Therefore, we investigated the TL regulation after multiple (chronic) UV-irradiations over weeks in a tissue context.
103 As mentioned earlier, chronic UV-exposed of NHEKs in OTCs showed an increase in TSI, which was not expected. So far, we cannot predict if this TSI increase indicated a real lengthening of telomeres or alternatively due to increased probe incorporation into the relaxed telomeric chromatin during qFISH staining as overserved in monolayer cultures. To enlighten the cause of the TSI increase further investigations including TL measurement by qPCR, would be necessary. Unfortunately, the extraction of telomeric DNA from OTCs for qPCR was not possible due to technical reasons. As increased ATM activation was seen after chronic UVA-irradiation in NHEK nuclei, the repair of this damage may lead to further DNA damage-dependent chromatin modulation in conjunction with increased probe binding to telomeres. On the contrary, a real increase in length could be due to telomerase activity. Indeed, Taylor and colleagues identified higher levels of telomerase activity in sun-exposed skin compared to non-exposed skin, suggesting that UV-light can increase telomerase activity (Taylor et al. 1996). Thus, both scenarios are possible and the discrimination between both may be possible by collecting additional data.
It is important to note that fibroblasts grown in co-cultures with NHEK (OTCs) showed different telomere length regulation than those grown in dermal equivalents without keratinocytes after irradiation with the same UVA doses. While normal human dermal fibroblasts (NHDFs) in OTCs showed increased TSIs after UVA-exposure, the TSIs of NHDFs in dermal equivalents was decreased.
Thus, it is possible that the presence of epidermal keratinocytes influenced the fibroblasts via cross-talk through soluble factors including cytokines (Bassino et al. 2017), impacting the telomere length regulation. Moreover, the presence of keratinocytes with their cornified layers may have also largely altered the UV dose ultimately reaching the NHDFs and thus, the response in telomere length. Thus, we concluded, that the telomere regulation of fibroblasts in UV-irradiated dermal equivalents is dependent on their environment in the model system.
Generally, telomere shortening as response to chronic UV-irradiation was most reliably reproduced in HaCaT cells within the OTC setting after UVA+B and even stronger after UVA-irradiation. As the repetitive telomeric DNA was found to be permissive for UV-induced damage in several publications (Clingen et al. 1995; Tommasi et al. 1997; Oikawa et al. 2001; Rochette & Brash 2010), here, we add the detection of telomeric DNA damage shortly after UV-irradiation in keratinocytes. Moreover, we propose that impairment of p53 that is present in HaCaT cells hinders telomeric DNA repair, likely as cells are not stopped in the cell cycle. As consequence, continued irradiation leads to the accumulation of damage that might lead to telomere erosion during cell cycle progression. This assumption is supported by the findings of a replication-dependent telomere shortening mechanism after the induction of single-strand breaks (SSB) through hydrogen peroxide. Thus, oxidative stress can lead to telomere shortening due to a specific repair deficiency during replication (Petersen et al. 1998; von Zglinicki et al. 2000). Furthermore, in an earlier study, I detected an increase of single-stranded binding protein protection of telomere-1 (POT-1) after UVA-irradiation at telomeres, which suggests the presence of UV-induced single-stranded breaks within the telomere sequence (Bort Master thesis
Discussion
104 2012). Taken together, UV-induces DNA damage especially at telomeres that cannot be repaired in HaCaT cells, as impaired p53 does not lead to the required growth arrest, causes telomere erosion over time.
Conclusion
Our data confirmed that stress whether acting endogenously or being induced by exogenous factors, influenced telomere length as shown for bariatric intervention and weight loss of obese patients causing a time-dependent telomere elongation in their PBMCs as well as for chronic UV-induced stress causing accelerated telomere erosion in p53-mutant skin keratinocytes. Importantly, and for the first time, this study additionally demonstrated that telomere length in PBMCs is not static but showed in as little as two-months-time intervals significant irregular oscillation/fluctuation. Thus, besides replication-dependent telomere loss, and loss under various stress conditions, as demonstrated in numerous recent studies, we encountered a continuous endogenous regulation of PBMC telomere length also in healthy probands. This regulation needs to be taken into account when considering telomere length as a biomarker. The mechanism of this native telomere length regulation, however, has not yet been fully understood.
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