Thomas Blatt

Vimentin Is the Specific Target in Skin Glycation STRUCTURAL PREREQUISITES, FUNCTIONAL CONSEQUENCES, AND ROLE IN SKIN AGING

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification Nϵ-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.

Krüppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.

Stiffening of Human Skin Fibroblasts with Age

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

A Circadian Clock in HaCaT Keratinocytes

To anticipate daily environmental changes, most organisms developed endogenous timing systems, the so-called circadian (∼24 hours) clocks. Circadian clocks exist in most peripheral tissues and govern a huge variety of cellular, metabolic, and physiological processes. Recent studies have suggested daytime-dependent variations in epidermal functions such as barrier recovery and pH homeostasis. However, a local circadian clock in epidermal keratinocytes has not been reported yet, and as such the molecular link between the circadian system and epidermal physiology remains elusive. In this study we describe a functional cell autonomous circadian clock in human adult low calcium temperature (HaCaT) keratinocytes. Using live-cell bioluminescence imaging and mRNA expression time series, we show robust circadian transcription of canonical clock genes in synchronized HaCaT keratinocytes. Genetic and pharmacological perturbation experiments as well as the phase relations between clock gene rhythms confirm that the molecular makeup of the HaCaT keratinocyte clock is very similar to that of other peripheral clocks. Furthermore, temperature was identified to be a potent time cue (Zeitgeber) for the epidermal oscillator. Temperature cycles entrain HaCaT keratinocytes, leading to the identification of rhythmic expression of several genes involved in epidermal physiology such as cholesterol homeostasis and differentiation. Thus, we present HaCaT keratinocytes as an excellent model to study the regulation of keratinocyte physiology by the circadian clock in a simple yet robust in vitro system.

[41] Topically applied antioxidants in skin protection

Publisher Summary Reactive oxygen species (ROS) and free radicals can be generated in skin by ultraviolet (UV) irradiation, ultrasonication, toxic or allergic chemical noxes, or even during normal metabolic processes of cells. More than 10 10 ROS are generated per day and per cell under normal physiological conditions, which have to be dealt with by the endogenous antioxidant system. Oxidative stress arises when the balance between antioxidant and prooxidant processes drifts in favor of a prooxidant status. Some of the general events in the early phase of oxidative stress response in skin are depletion of endogenous intra- and intercellular antioxidants, the enhancement of the intracellular hydroperoxide level, and the induction of specific signal transduction pathways. This chapter presents an example of a screening strategy for the successful selection of in vivo functional topical antioxidants using special in vitro, ex vivo, and in vivo methods. The screening protocol used is subdivided into three steps. In the primary step, the efficacy of antioxidants is evaluated in vitro in cultures of primary skin cells, and the biocompatibility of the selected efficient antioxidants is proved in vitro and in vivo. In the second step, the antioxidant efficacy is tested ex vivo in a clinical pilot study using biopsies from in vivo -pretreated skin sites. In the third step, the in vivo efficacy of preselected antioxidants has been confirmed in clinical studies on panels of human volunteers.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

MODULATION DES OXIDATIVEN STRESSES IN DER HUMANEN ALTERSHAUT

Zusammenfassung Oxidativer Streß (UV-Licht, freie Radikale) ist einer der wesentlichen Auslöser für vorzeitige Hautalterung. Als aktive Schutzmechanismen gegen diese oxidativen Schäden, die besonders im Alter zunehmen, können Koenzym Q10 (CoQ10), aber auch exogen applizierte Antioxidanzien wirken.Unsere vergleichenden In-vitro- und In-vivo-Untersuchungen an Haut alter Probanden zeigen anhand der Meßparameter (ultraschwache Photonenemission, Gesamtthiolstatus, Mitochondrienmembranpotential und Zellvitalität), daß die endogene Resistenz gegen UV-Licht in Keratinozyten alter Spender reduziert ist. Diese geringere Resistenz, d.h. der schlechtere Schutz der epidermalen Zellen gegen oxidative Stressoren, insbesondere gegen UV-Licht, kann durch topische Applikation von Verbindungen wie CoQ10 und Antioxidanzien wie alpha-Glucosylrutin (15) deutlich verbessert werden. Placebokontrollierte In-vivo-Studien zeigen außerdem, daß bereits vorhandene, vornehmlich durch Lichtalterung (Photoaging) bedingte Hautveränderungen, wie z.B. sichtbare Fältchen im Bereich der Augenwinkel, durch topische Langzeitbehandlung mit humanidentischem CoQ10 deutlich reduziert werden können.Summary Oxidative stress (UV irradiation, free radicals) plays a significant role in aging. Coenzyme Q10 (CoQ10) and exogenously applied antioxidants can significantly reduce the formation of oxidative stress with increasing age.In our in vitro and in vivo experiments concerning the parameters of ultraweak photon emission (UPE), intracellular thiol status, mitochondrial membrane potential and cell vitality, we demonstrated a diminished resistance in keratinocytes of old donors against UV irradiation. This reduced epidermal resistance against oxidative stressors, i.e. UV irradiation, can be improved by topical application of CoQ10 and antioxidants like alpha-glucosylrutin (15). Furthermore, our in vivo investigations show that wrinkles around the region of the eyes (“crow feet”) could be reduced by long-term application of CoQ10.

Effects of Glyceryl Glucoside on AQP3 Expression, Barrier Function and Hydration of Human Skin

Background/Aim: Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. Methods: In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. Results: AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. Conclusion: GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin.

Biochemical rationale and experimental data on the antiaging properties of CoQ10 at skin level

Coenzyme Q(10) (CoQ(10) ) is a key component of the mitochondrial respiratory chain and, therefore, is essential for the bioenergetics of oxidative phosphorylation. It is also endowed with antioxidant properties, and recent studies pointed out its capability of affecting the expression of different genes. In this review, we analyze the data on the mechanisms by which CoQ(10) interacts with skin aging processes. The effect of CoQ(10) in preserving mitochondrial function cooperates in maintaining a proper energy level, which serves to prevent the aging skin from switching to anaerobic energy production mechanisms. Furthermore, the antioxidant capacity of CoQ(10) contributes to a positive effect against UV-mediated oxidative stress. Some of these effects have been assessed also in vivo, by the sensitive technique of ultraweak photoemission. Finally, CoQ(10) has been shown to influence, through a gene induction mechanism, the synthesis of some key proteins of the skin and to decrease the expression of some metalloproteinase such as collagenase. These mechanisms may also contribute to preserve collagen content of the skin.

Folic acid and creatine improve the firmness of human skin in vivo

Background  The decrease in firmness is a hallmark of skin aging. Accelerated by chronic sun exposure, fundamental changes occur within the dermal extracellular matrix over the years, mainly impairing the collagenous network.