A NEW APPROACH FOR SKIN CARE RESETTING CUTANEOUS CELLS' INTERNAL CLOCK

DO_1284GB1006A

A NEW APPROACH FOR SKIN CARE : RESETTING CUTANEOUS CELLS INTERNAL CLOCK

Keywords : circadian rhythms, clock genes, temporal resetting, sunlight mimic Short abstract: this article describes the mechanisms responsible for the synchronization of cutaneous cells metabolism with the circadian rhythm. A new approach for skin care, consisting in favouring this synchronization, is presented.

A large number of physiological processes (heartbeat and blood flow, body temperature, renal activity, liver metabolism) are regulated by the circadian cycle, the periodic succession of day and night. As a matter of fact, synchronization of the organism with this environmental oscillation is of utmost importance for health, and even survival.1 The skin, more than any other organ, requires a tight control of its rhythmic biological functions because it is directly exposed to high amounts of light. In agreement with this, many important functions of the skin such as eccrine sweating, epidermal cell proliferation, sebum excretion, and barrier function fluctuate over a 24 hours period.2,3As a general consideration, the skin logically promotes its protective functions during the day (the challenging phase), and favours the regenerative processes during the night (the recovering phase). Many reports have described how this may govern topical treatments efficacy or side-effects.4,5 Recent advances in the understanding of the molecular mechanisms responsible for the temporal programming of the skin open new horizons for cosmetic treatments. Improvement of the temporal organization of some skin variables appears as a new attractive issue for skin care. Mechanisms driving biological rhythmicity Two separate mechanisms participate to the setting and the maintenance of a synchronized biological rhythmicity. Sunlight is of course the major synchronizing signal. Activation of photoreceptors in the retina, and transmission to the central nervous system via
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the retinohypothalamic tract, triggers neural outputs and endocrine secretions that will synchronize the peripheral organs. The second mechanism, independent from the other, is an endogenous timekeeping mechanism, usually named circadian clock or biorhythm. It is a cell autonomous process involving several intra-cellular proteins engaged in auto-regulatory loops. More than 80 genes with clear rhythmic expression, some of them called circadian clock genes, participate to this clock-in-cell process. These genes seem to regulate most of the cell molecular machinery.6 This secondary internal mechanism probably helps adaptive systems to anticipate the oscillation, but one consequence is a continuous need for resetting of the internal clock. The endogenous cellular oscillator must follow environmental fluctuation such as seasonal changes, for instance. Consequences of synchronicity disruption between these two mechanisms are experienced with daylight savings, jet lag or late age. Skin specificity in the maintenance of synchronicity Peripheral tissues, including cutaneous tissues, have intra-cellular oscillators and thereby have their own internal clock. Keratinocytes and fibroblasts express several circadian clock genes.
7

Thus, the skin may theoretically

experience synchronicity disruption, with possible negative consequences on its rhythmic biological functions. Synchronization is of particular concern for this organ. This is illustrated, for instance, by the fact that in the newborn the rhythmicity appears first in the cutaneous tissues. It seems that for this reason the skin has a specific resetting mechanism that superimposes to the retinadriven resetting mechanism. Several in vivo and in vitro experimental evidences suggest that the skin behaves as an extraretinal photoreceptor, that is to say that light can directly (independently from the visual pathway) entrain the circadian internal clock of cutaneous cells. Some blind patients maintain a circadian rhythm of plasma melatonin concentration, and it was shown that light exposure restricted to parts of the body (no light reaches the retina), can modify some circadian biochemical processes.8 The slight rhythmicity shift observed between facial skin and body skin (non exposed to sunlight),9 is also indicative of a direct effect of light on skin. It was shown recently that ultraviolet B radiations (UVB)
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can induce a rhythmic expression of circadian clock genes in cultured keratinocytes.10 According to the authors, the ability of UVB to reset cutaneous circadian rhythms would rely on the redox modulation of signal transduction. This set of data suggests that skin tissues may experience limited synchronicity disruption (retina-driven mechanisms remain the main pacemaker), due to inadequate activation of the specific resetting mechanism. These findings prompted us to investigate the effect of a chemical sunlight mimic (GAEI), able to reproduce some effects of low-doses UVB radiations (redox modulation), on epidermal cells circadian clock. For this, we have designed an original experimental model. In vitro study of rhythmic processes in the epidermis Human reconstituted epidermis (HRE), a 3-D in vitro model obtained from normal keratinocytes that presents high histo-morphological and biochemical homologies with normal epidermis, were exposed to low doses of solar simulated radiations (SSR : wavelengths between 250 and 800 nm), or incubated with the sunlight mimic compound GAEI (Figure 1).

Sunlight mimic
(GAEI)

OR

Solar Simulated Radiations (SSR)

Cell viabilitygenotoxicity assessments

Oscillating biological functions

Cross-checking MTT & LDH assays + -actin gene expression

Cellular oscillators (clock-in-the-cell)

Cell proliferation vit. D3 metabolism

1
Circadian clock genes expression

Figure 1. Experimental design for the monitoring of rhythmic processes within the epidermis
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Several endpoints were monitored following exposure to the stimuli (SSR or sunlight mimic): 1. Assessment of cell viability and absence of genotoxicity A reliable monitoring of rhythmic processes within the HRE requires the demonstration that both stimuli do not induce cell toxicity or genotoxicity. Cell viability was assessed by cross-checking with two techniques (MTT and LDH release assays). Cell mortality generated by the irradiation was very limited, and thus cannot interfere with circadian processes. A representative example of cell viability variations following SSR exposure is reported in table 1.

Incubation time after irradiation (hours)

LDH activity in the MTT assay : cell viability reported to the non- culture medium (nmol of NADH.min-1.ml-1) irradiated control (%)

1 6 14 24 35

88 78 84 93 72

0,57 1,14 N.D. 0,47 0,60

Table 1. Cross-checking of the cell viability in HRE exposed to SSR (2 kJ.m-2) with the MTT and LDH release assays. (N.D. : not detected). No significant variations of cell viability were observed in HRE incubated with the sunlight-mimic GAEI (data not shown). Innocuity of this active ingredient had already been verified in similar experimental conditions.11 Absence of major DNA damage was anticipated regarding the SSR doses applied. Histological study after haematoxylin-eosine staining confirms absence of apoptotic cells (cells with pycnotic nuclei and intensely eosinophilic cytoplasm) following exposure to SSR or GAEI. In addition, the expression level of the reference non circadian beta-actin gene, which is constitutively expressed in cutaneous cells (this is a housekeeping gene), was monitored using a semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) technique. The stimuli, SSR or GAEI, didnt induce significant variations of the expression level of the
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housekeeping gene. This accounts for a limited impact on the genome expression. 2. Assessment of circadian clock genes expression Expression levels of the two circadian clock genes per-1 and clock, encoding for transcription factors (PER and CLOCK) involved in the internal clock setting,7 were measured by semi-quantitative RT-PCR. In our 3-D in vitro model per-1 and clock expression was detected by electrophoresis after amplification (Figure 2), and was relatively constant in the absence of any stimulus. This had been previously reported for 2-D human keratinocytes cultures.7,10

Figure 2. Circadian clock genes expression in human reconstituted epidermis. Detection of PCR products. Lane 1, DNA molecular weight ladder ; lane 2, beta-actin (538pb) ; lane 3, clock (488pb); lane 4, per-1 (382pb) 3. Assessment of biological functions oscillation Epidermal cell multiplication was assessed with the immuno-histochemical detection of the KI67 protein expression within the HRE. Complementary measurements of transcriptional activity (total RNA synthesis) and translational activity (total protein synthesis) were carried out in parallel. Vitamin D metabolism was also investigated. A preliminary experiment consisted in the assessment of the photo-transformation of the vitamin precursor 7-dehydrocholesterol (7-DHC) into pre-vitamin D3 (pre-VD3) using the HPLC method described by B. Lehmann and coworkers.12 In our experimental conditions we found that limited but still measurable amounts of pre-VD3 were formed within the epidermis following irradiation with SSR (figure 3).

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Figure 3. HPLC monitoring of 7-dehydrocholesterol (7-DHC) conversion into previtamin D3 (pre-VD3), following HRE exposure to SSR (2 kJ.m-2). We have then determined by semi-quantitative RT-PCR the expression level of the gene encoding for 25OHD-1alpha hydroxylase (1OHase), the enzyme that transforms pre-VD3 into the important regulator of epidermal differentiation dihydroxyvitamin D (calcitriol).13,14 Experimental results 1. SSR-induced oscillations of circadian clock genes Circadian clock genes per-1 and clock expression levels were monitored over a 35 hours period of time following irradiation with SSR (2 kJ.m-2). For more reliable measurements, expression levels of clock genes were reported to the (non variable) beta-actin gene expression for each time points. Relative expression levels correspond to : [circadian gene/beta-actin]irradiated / [circadian gene/beta-actin]non-irradiated

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Relative units
2,5 2 1,5 1 0,5 0 0 10 20 30 Clock Per-1

Time (hours)
40

Figure 4. Relative expression levels of the circadian clock genes, estimated by semiquantitative RT-PCR Circadian clock genes responses to SSR peak at about 14 hours after irradiation, and falls back to baseline values after about 24h. Variations were more significant for per-1 than for clock. Interestingly, measurements 35 hours after irradiation suggest that SSR induce a rhythmic expression of per-1 and clock. Basal expression levels and variation amplitudes may vary a lot from one experiment to another, but the general profile remains, with a periodicity of about 24 hours. 2. Effects of the sunlight mimic GAEI on clock genes Glutamylamidoethylimidazole (GAEI)a is a peptidomimetic designed for improved cutaneous penetration and resistance to enzymatic deactivation (figure 5). Applied in small quantities on the epidermis (dosage in the micromolar range), GAEI is able to produce non-toxic very low amounts of the redox-active superoxide anion.15 It is hypothesized that low amounts of reactive species derived from oxygen (ROS) are the effectors for circadian gene expression modulation by sunlight.10 GAEI cannot induce an overproduction of ROS since at higher concentration (at the millimolar range), it behaves as an antioxidant.15

H2 N HOOC

H NH O N NH

Figure 5. Chemical structure of glutamylamidoethylimidazole (GAEI), a sunlight mimic Footnote a): GAEI is a patented product commercialised under the name of CHRONOCYCLIN, a registered trade name.
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HRE were incubated with GAEI (50 microM) in the absence of any light, and both circadian clock genes expression profiles were recorded. Kinetics curves were very similar to the ones obtained after irradiation with SSR (figure 6 and 7).

Relative units
2,5 2 1,5 1 0,5 0 0 10 20 30 GAEI SRR

Time (hours)
40

Figure 6. Per-1 relative gene expression after exposure to SSR (2 kJ.m-2) or GAEI (50 microM), estimated by semi-quantitative RT-PCR
Relative units
5 4 3 2 1 0 0 10 20 30 40 GAEI SSR

Time (hours)

Figure 7. Clock relative gene expression after exposure to SSR (2 kJ.m-2) or GAEI (50 microM) estimated by semi-quantitative RT-PCR 3. Biological oscillations following exposure to the stimuli The exact role of the proteins encoded by per-1 and clock (PER and CLOCK) is still unclear. However, their nuclear location7 and their expression at some specific phases of the cell-cycle suggest their involvement in cell proliferation control. Attempts to correlate clock gene RT-PCR data with increased transcriptional (quantification of total RNA extracted
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from

HRE),

or

translational

(quantification of total proteins) activities were unsuccessful. Immunostaining

of the KI67 protein in HRE has given some results, however, a lack of reproducibility with this technique impedes an effective correlation. An indicative result is reported in figure 8.

Figure 8. KI67 protein expression time course in HRE treated with the sunlight mimic GAEI. Correlation of circadian clock genes variations with vitamin D metabolization, another light-driven biochemical process, was possible. Following exposure of HRE to SSR or the sunlight mimic GAEI, expression of the gene that encodes for 1OHase, the enzyme that catalyses the final biotransformation of a vitamin D intermediate into calcitriol, tends to become rhythmic and followed roughly the kinetics of clock and per-1 (figure 9).

Relative units
4 3,5 3 2,5 2 1,5 1 0,5 0 0 20 40
GAEI SSR

Time (hours)

Figure 9. Expression levels of the gene encoding for 25OHD-1alpha hydroxylase (1OHase) in HRE exposed to SSR or treated with the sunlight mimicGAEI

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Conclusion We confirm that non toxic low doses of solar radiations modulate the expression of circadian clock genes in epidermal cells. Our results account for a direct resetting mechanism of the internal clock of epidermal cells that may participate to metabolic settings needed for diurnal conditions. In our experimental conditions, the sunlight mimic GAEI, a compound able to reproduce some effects of low-doses UVB radiations (redox modulation), could also reset circadian clock genes in a very similar way than simulated solar radiations. We provide some evidences that clock genes resetting may result in an optimized barrier function. Epidermal vitamin D metabolism, which is involved in the maintenance of the permeability barrier homeostasis13, seems to be controlled by the clock gene machinery. Modern lifestyle may be responsible for synchronicity disruption between our internal clock and the environmental time. Physiological discomfort experienced with jet lag is the most well known consequence, but night-time work and limited sleeping time can also generate pathological states associated with desynchronization. Chronic disruption is also associated with aging.16 Synchronicity disruption is likely to occur in the skin too, and since a strict concordance between the internal oscillation and the environmental oscillation seems to be necessary for this organ, this may have severe consequences (weakened barrier function, dehydration, impaired defence systems). Therefore, the sunlight mimic GAEI may generate a wake-up signal that will help the skin to better adapt to circadian changes.

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