Different immortalized keratinocyte cell lines display distinct capabilities to differentiate and reconstitute an epidermis in vitro.

Dermatological research relies on the availability of suitable models that most accurately reflect the in vivo situation. Primary keratinocytes obtained from skin reduction surgeries are not only limited by availability but have a short lifespan and show donor-specific variations, which hamper the understanding of general mechanisms. The spontaneously immortalized keratinocyte cell line HaCaT displays chromosomal aberrations and is known to differentiate in an abnormal manner. To overcome these issues, we validated different engineered immortalized cell lines created from primary human keratinocytes (NHK) as model systems to study epidermal function. Cell lines either immortalized by the expression of SV40 large T antigen and hTERT (NHK-SV/TERT) or by transduction with HPV E6/E7 (NHK-E6/E7) were analysed for their growth and differentiation behaviour using 2D and 3D culture systems and compared to primary keratinocytes. Both cell lines displayed a robust proliferative behaviour but were still sensitive to contact inhibition. NHK-E6/E7 could be driven into differentiation by Ca2+ switch, while NHK-SV/TERT needed withdrawal from any proliferative signal to initiate a delayed onset of differentiation. In 3D epidermal models both cell lines were able to reconstitute a stratified epidermis and functional epidermal barrier. However, only NHK-E6/E7 showed a degree of epidermal maturation and stratification that was comparable to primary keratinocytes.

The PI3K-Akt-mTOR and Associated Signaling Pathways as Molecular Drivers of Immune-Mediated Inflammatory Skin Diseases: Update on Therapeutic Strategy Using Natural and Synthetic Compounds.

The dysregulated phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway has been implicated in various immune-mediated inflammatory and hyperproliferative dermatoses such as acne, atopic dermatitis, alopecia, psoriasis, wounds, and vitiligo, and is associated with poor treatment outcomes. Improved comprehension of the consequences of the dysregulated PI3K/Akt/mTOR pathway in patients with inflammatory dermatoses has resulted in the development of novel therapeutic approaches. Nonetheless, more studies are necessary to validate the regulatory role of this pathway and to create more effective preventive and treatment methods for a wide range of inflammatory skin diseases. Several studies have revealed that certain natural products and synthetic compounds can obstruct the expression/activity of PI3K/Akt/mTOR, underscoring their potential in managing common and persistent skin inflammatory disorders. This review summarizes recent advances in understanding the role of the activated PI3K/Akt/mTOR pathway and associated components in immune-mediated inflammatory dermatoses and discusses the potential of bioactive natural products, synthetic scaffolds, and biologic agents in their prevention and treatment. However, further research is necessary to validate the regulatory role of this pathway and develop more effective therapies for inflammatory skin disorders.

The Ca2+ channel TRPV4 is dispensable for Ca2+ influx and cell volume regulation during hypotonic stress response in human keratinocyte cell lines.

Cell swelling as a result of hypotonic stress is counteracted in mammalian cells by a process called regulatory volume decrease (RVD). We have recently discovered that RVD of human keratinocytes requires the LRRC8 volume-regulated anion channel (VRAC) and that Ca2+ exerts a modulatory function on RVD. However, the ion channel that is responsible for Ca2+ influx remains unknown. We investigated in this study whether the Ca2+-permeable TRPV4 ion channel, which functions as cell volume sensor in many cell types, may be involved in cell volume regulation during hypotonic stress response of human keratinocytes. We interfered with TRPV4 function in two human keratinocyte cell lines (HaCaT and NHEK-E6/E7) by using two TRPV4-specific inhibitors (RN1734 and GSK2193874), and by creating a CRISPR/Cas9-mediated genetic TRPV4-/- knockout in HaCaT cells. We employed electrophysiological patch clamp analysis, fluorescence-based Ca2+ imaging and cell volume measurements to determine the functional importance of TRPV4. We could show that both hypotonic stress and direct activation of TRPV4 by the specific agonist GSK1016790A triggered intracellular Ca2+ response. Strikingly, the Ca2+ increase upon hypotonic stress was neither affected by genetic knockout of TRPV4 in HaCaT cells nor by pharmacological inhibition of TRPV4 in both keratinocyte cell lines. Accordingly, hypotonicity-induced cell swelling, downstream activation of VRAC currents as well as subsequent RVD were unaffected both in TRPV4 inhibitor-treated keratinocytes and in HaCaT-TRPV4-/- cells. In summary, our study shows that keratinocytes do not require TRPV4 for coping with hypotonic stress, which implies the involvement of other, yet unidentified Ca2+ channels.

mTORC1 Activity in Psoriatic Lesions Is Mediated by Aberrant Regulation through the Tuberous Sclerosis Complex.

In the basal, proliferative layer of healthy skin, the mTOR complex 1 (mTORC1) is activated, thus regulating proliferation while preventing differentiation. When cells leave the proliferative, basal compartment, mTORC1 signaling is turned off, which allows differentiation. Under inflammatory conditions, this switch is hijacked by cytokines and prevents proper differentiation. It is currently unknown how mTORC1 is regulated to mediate these effects on keratinocyte differentiation. In other tissues, mTORC1 activity is controlled through various pathways via the tuberous sclerosis complex (TSC). Thus, we investigated whether the TS complex is regulated by proinflammatory cytokines and contributes to the pathogenesis of psoriasis. TNF-α as well as IL-1ß induced the phosphorylation of TSC2, especially on S939 via the PI3-K/AKT and MAPK pathway. Surprisingly, increased TSC2 phosphorylation could not be detected in psoriasis patients. Instead, TSC2 was strongly downregulated in lesional psoriatic skin compared to non-lesional skin of the same patients or healthy skin. In vitro inflammatory cytokines induced dissociation of TSC2 from the lysosome, followed by destabilization of the TS complex and degradation. Thus, we assume that in psoriasis, inflammatory cytokines induce strong TSC2 phosphorylation, which in turn leads to its degradation. Consequently, chronic mTORC1 activity impairs ordered keratinocyte differentiation and contributes to the phenotypical changes seen in the psoriatic epidermis.

Mechanism of anti-inflammatory effects of rifampicin in an ex vivo culture system of hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease of the hair follicles leading to painful lesions, associated with increased levels of pro-inflammatory cytokines. Numerous guidelines recommend antibiotics like clindamycin and rifampicin in combination, as first-line systemic therapy in moderate-to-severe forms of inflammation. HS has been proposed to be mainly an auto-inflammatory disease associated with but not initially provoked by bacteria. Therefore, it has to be assumed that the pro-inflammatory milieu previously observed in HS skin is not solely dampened by the bacteriostatic inhibition of DNA-dependent RNA polymerase. To further clarify the mechanism of anti-inflammatory effects of rifampicin, ex vivo explants of lesional HS from 8 HS patients were treated with rifampicin, and its effect on cytokine production, immune cells as well as the expression of Toll-like receptor 2 (TLR2) were investigated. Analysis of cell culture medium of rifampicin-treated HS explants revealed an anti-inflammatory effect of rifampicin that significantly inhibiting interleukin (IL)-1ß, IL-6, IL-8, IL-10 and tumour necrosis factor (TNF)-α production. Immunohistochemistry of the rifampicin-treated explants suggested a tendency for it to reduce the expression of TLR2 while not affecting the number of immune cells.

Cell Volume Regulation in the Epidermis.

In order to cope with external stressors such as changes in humidity and temperature or irritating substances, the epidermis as the outermost skin layer forms a continuously renewing and ideally intact protective barrier. Under certain circumstances, this barrier can be impaired and epidermal cells have to counteract cell swelling or shrinkage induced by osmotic stress via regulatory volume decrease (RVD) or increase (RVI). Here, we will review the current knowledge regarding the molecular machinery underlying RVD and RVI in the epidermis. Furthermore, we will discuss the current understanding how cell volume changes and its regulators are associated with epidermal renewal and barrier formation.

IL-17E (IL-25) and IL-17A Differentially Affect the Functions of Human Keratinocytes.

Our group has recently shown that keratinocyte-derived IL-17E (IL-25), one of six members of the IL-17 family, is overexpressed in lesional psoriatic skin and is involved in its pathophysiology. We show here that IL-22 enhances IL-17E production in human keratinocytes and that these cells display a complete IL-17E receptor at their surface, the expression of which is further induced by IL-17A, indicating a potential autocrine effect of IL-17E. Therefore, we addressed the impact of IL-17E on the function of human primary keratinocytes. IL-17E promoted the proliferation of keratinocytes in two-dimensional and three-dimensional cultures and caused the concomitant upregulation of differentiation-associated gene transcripts (e.g., keratin 10), whereas their expression was either inhibited or not changed by IL-17A. Contrary to IL-17A, IL-17E was not involved in the induction of antimicrobial proteins. Time-lapse analysis of cell movement showed that IL-17E influences cell motility, increasing both cell speed and displacement. This was associated with specific changes in the actin cytoskeleton organization and the cell-substrate adhesion. No such effects were observed upon IL-17A stimulation. In summary, we identified effects of IL-17E clearly distinct from IL-17A, pointing toward an important role of IL-17E in the physiology and pathophysiology of the epidermis.

Epidermal mTORC1 Signaling Contributes to the Pathogenesis of Psoriasis and Could Serve as a Therapeutic Target.

Although modern biologics targeting different inflammatory mediators show promising therapeutic success, comprehensive knowledge about the molecular events in psoriatic keratinocytes that contribute to the pathogenesis and could serve as therapeutic targets is still scarce. However, recent efforts to understand the deregulated signal transduction pathways have led to the development of small molecule inhibitors e.g., tofacitinib targeting the Jak/Stat cascade that opens additional therapeutic options. Recently, the PI3-K/Akt/mTOR signaling pathway has emerged as an important player in the control of epidermal homeostasis. This review summarizes the current knowledge on the role of this pathway in the pathogenesis of psoriasis, especially the epidermal manifestation of the disease and discusses current approaches to target the pathway therapeutically.

Hypotonic stress response of human keratinocytes involves LRRC8A as component of volume-regulated anion channels.

The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume-regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT- and NHEK-LRRC8A knockout cells by using CRISPR-Cas9. I- influx assays using halide-sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein-AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes.

Inflammation dependent mTORC1 signaling interferes with the switch from keratinocyte proliferation to differentiation.

Psoriasis is a frequent and often severe inflammatory skin disease, characterized by altered epidermal homeostasis. Since we found previously that Akt/mTOR signaling is hyperactivated in psoriatic skin, we aimed at elucidating the role of aberrant mTORC1 signaling in this disease. We found that under healthy conditions mTOR signaling was shut off when keratinocytes switch from proliferation to terminal differentiation. Inflammatory cytokines (IL-1ß, IL-17A, TNF-α) induced aberrant mTOR activity which led to enhanced proliferation and reduced expression of differentiation markers. Conversely, regular differentiation could be restored if mTORC1 signaling was blocked. In mice, activation of mTOR through the agonist MHY1485 also led to aberrant epidermal organization and involucrin distribution. In summary, these results not only identify mTORC1 as an important signal integrator pivotal for the cells fate to either proliferate or differentiate, but emphasize the role of inflammation-dependent mTOR activation as a psoriatic pathomechanism.

Endothelial cells are highly heterogeneous at the level of cytokine-induced insulin resistance.

The endothelial wall plays a crucial role in various diseases as it serves as the barrier between circulatory system and organ tissue. Inflammation-driven insulin resistance and subsequent endothelial dysfunction represent a pathomechanism in cardiovascular diseases such as atherosclerosis and myocardial infarction. It was recently suggested that insulin resistance also contributes to the pathogenesis of psoriasis, a chronic inflammatory disease of the skin. However, it is not clear whether similar mechanisms at the endothelium contribute to the disease. In this study, we ask which endothelial cells are most suitable to address this question. We investigated the insulin response of four cell types (primary cells and cell lines) representing different vascular beds (micro- and macrovascular cells) in the presence of different pro-inflammatory cytokines. All four cell types used responded well to insulin; however, the ability to become resistant to insulin due to an inflammatory stimulus by cytokines involved in psoriasis (e.g. IL-1ß, IL-12, IL-17, IL-23 and TNF-α) was very heterogeneous and could not be attributed to the differential expression of the cognate cytokine receptors. We conclude that this disparity is due to the different origins and properties of the endothelial cells used. Thus, endothelial cells should be carefully selected for the purpose of the respective study, particularly when it comes to analysing the pathogenesis of a disease and the search of new molecular targets for innovative therapies.

Interleukin-1ß interferes with epidermal homeostasis through induction of insulin resistance: implications for psoriasis pathogenesis.

Response pathways of the metabolic and the immune system have been evolutionary conserved, resulting in a high degree of integrated regulation. Insulin is a central player in the metabolic system and potentially also in the homeostasis of the skin. Psoriasis is a frequent and often severe autoimmune skin disease, clinically characterized by altered epidermal homeostasis, of which the molecular pathomechanisms are only little understood. In this study, we have examined a potential role for insulin signaling in the pathogenesis of this disease. We show that IL-1ß is present in high quantities in tissue fluid collected via microdialysis from patients with psoriasis; these levels are reduced under successful anti-psoriatic therapy. Our results suggest that IL-1ß contributes to the disease by dual effects. First, it induces insulin resistance through p38MAPK (mitogen-activated protein kinase), which blocks insulin-dependent differentiation of keratinocytes, and at the same time IL-1ß drives proliferation of keratinocytes, both being hallmarks of psoriasis. Taken together, our findings point toward insulin resistance as a contributing mechanism to the development of psoriasis; this not only drives cardiovascular comorbidities, but also its cutaneous phenotype. Key cytokines inducing insulin resistance in keratinocytes and kinases mediating their effects may represent attractive targets for novel anti-psoriatic therapies.

Characterization of the intrinsic and TSC2-GAP-regulated GTPase activity of Rheb by real-time NMR.

Tuberous sclerosis complex 2 (TSC2), whose gene is frequently mutated in tuberous sclerosis, increases the guanosine triphosphatase (GTPase) activity of the small heterotrimeric GTP-binding protein (G protein) Rheb, thus resulting in the decreased activity of the mammalian target of rapamycin (mTOR), the master regulator of cell growth. Here, we describe the development of a nuclear magnetic resonance (NMR)-based, quantitative, real-time assay to explore the molecular mechanism of the intrinsic and TSC2-catalyzed GTPase activity of Rheb. We confirmed that TSC2 accelerated GTP hydrolysis by Rheb 50-fold through an "asparagine-thumb" mechanism to substitute for the nonfunctional "catalytic" glutamine of Rheb and we determined that catalysis was enthalpy driven. Most, but not all, of the disease-associated GTPase-activating protein (GAP) domain mutants of TSC2 that we examined affected its enzymatic activity. This method can now be applied to study the function and regulation of other GTPases.

Peptide aptamers with binding specificity for the intracellular domain of the ErbB2 receptor interfere with AKT signaling and sensitize breast cancer cells to Taxol.

The ErbB2 receptor tyrosine kinase is overexpressed in approximately 30% of breast tumor cases and its overexpression correlates with an unfavorable prognosis. A major contributor for this course of the disease is the insensitivity of these tumors toward chemotherapy. Monoclonal antibodies, inhibiting the ligand-induced activation of the receptor and tyrosine kinase inhibitors acting on the intrinsic enzymatic activity of the intracellular domain, have been developed as targeted drugs. Both have been shown to be beneficial for breast cancer patients. We targeted a third aspect of receptor function: its association with intracellular signaling components. For this purpose, we selected peptide aptamers, which specifically interact with defined domains of the intracellular part of the receptor. The peptide aptamers were selected from a random peptide library using a yeast two-hybrid system with the intracellular tyrosine kinase domain of ErbB2 as a bait construct. The peptide aptamer AII-7 interacts with high specificity with the ErbB2 receptor in vitro and in vivo. The aptamers colocalized with the intracellular domain of ErbB2 within cells. We investigated the functional consequences of the aptamer interaction with the ErbB2 receptor within tumor cells. The aptamer sequences were either expressed intracellularly or introduced into the cells as recombinant aptamer proteins. The phosphorylation of p42/44 mitogen-activated protein kinase was nearly unaffected and the activation of signal transducers and activators of transcription-3 was only modestly reduced. In contrast, they strongly inhibited the induction of AKT kinase in MCF7 breast cancer cells treated with heregulin, whereas AKT activation downstream of insulin-like growth factor I or epidermal growth factor receptor was not or only slightly affected. High AKT activity is responsible for the enhanced resistance of ErbB2-overexpressing cancer cells toward chemotherapeutic agents. Peptide aptamer interference with AKT activation resulted in the restoration of regular sensitivity of breast cancer cells toward Taxol.

Localization of Rheb to the endomembrane is critical for its signaling function.

Rheb, a small GTPase, has emerged as a key molecular switch that directly regulates the activity of the mammalian target of rapamycin (mTOR). Similar to other members of the Ras superfamily, Rheb has a C-terminal CaaX box that is subject to farnesylation. This study reports that farnesylation is a key determinant of Rheb's subcellular localization and directs its association with the endomembrane. Timed imaging of live cells expressing EGFP-Rheb reveals that following brief association with the ER, Rheb localizes to highly ordered, distinct structures within the cytoplasm that display characteristics of Golgi membranes. Failure of Rheb to localize to the endomembrane impairs its ability to interact with mTOR and activate downstream targets. Consistent with the notion that the endomembrane may serve as a platform for the assembly of a functional Rheb/mTOR complex, treatment of cells with brefeldin A interferes with transmission of Rheb signals to p70S6K.

The interaction of specific peptide aptamers with the DNA binding domain and the dimerization domain of the transcription factor Stat3 inhibits transactivation and induces apoptosis in tumor cells.

The transcription factor signal transducer and activator of transcription (Stat) 3 is activated through the interleukin-6 family of cytokines and by binding of growth factors to the epidermal growth factor (EGF) receptor. It plays an essential role in embryonic development and assumes specialized tasks in many differentiated tissues. Constitutively activated Stat3 has been found in tumor cell lines and primary tumors and plays a crucial role in tumor cell survival and proliferation. To inhibit the oncogenic action of Stat3 in tumor cells, we have selected short peptides, so-called peptide aptamers, which specifically interact with defined functional domains of this transcription factor. The peptide aptamers were selected from a peptide library of high complexity by an adaptation of the yeast two-hybrid procedure. Peptide aptamers specifically interacting with the Stat3 dimerization domain caused inhibition of DNA binding activity and suppression of transactivation by Stat3 in EGF-responsive cells. Similarly, a peptide aptamer selected for its ability to recognize the Stat3 DNA binding domain inhibited DNA binding and transactivation by Stat3 following EGF stimulation of cells. Peptide aptamers were expressed in bacteria as fusion proteins with a protein transduction domain and introduced into human myeloma cells. This resulted in dose-dependent growth inhibition, down-regulation of Bcl-x(L) expression, and induction of apoptosis. The inhibition of Stat3 functions through the interaction with peptide aptamers counteracts the transformed phenotype and could become useful in targeted tumor therapy.

Bifunctional recombinant proteins in cancer therapy: cell penetrating peptide aptamers as inhibitors of growth factor signaling.

The major aim of molecular cancer research is the development of new therapeutic strategies and compounds that target directly the genetic and biochemical causes of malignant transformation. Therapeutic genes, antibodies and their derivatives, but also small molecular weight compounds, have been used for this purpose. Small peptides might be able to complement these agents because of their ability to recognize specific protein domains and thus to interfere with enzymatic functions or protein-protein interactions. A variation of the yeast-two-hybrid procedure allows to select specifically binding peptides, so called peptide aptamers, from a peptide library of high complexity. This selection procedure can be adapted to any protein or protein fragment as a bait construct and selects for the intracellular interaction between the bait of choice and the peptide aptamer prey. Peptide aptamers thus selected can be cloned, provided with a protein transduction domain, expressed in bacteria and introduced into cancer cells. There they might disrupt protein-protein interactions crucial for cancer cell growth or survival. We introduce an example in which the Stat3 arm of EGF receptor signaling is selectively inhibited by a peptide aptamer and causes the growth arrest of EGF receptor-dependent tumor cells. The aptamer constructs can be supplemented with additional functional domains to enhance their inhibitory effects.

Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells.

Receptor tyrosine kinases of the epidermal growth factor (EGF) receptor family regulate essential cellular functions such as proliferation, survival, migration, and differentiation but also play central roles in the etiology and progression of tumors. We have identified short peptide sequences from a random peptide library integrated into the thioredoxin scaffold protein, which specifically bind to the intracellular domain of the EGF receptor (EGFR). These molecules have the potential to selectively inhibit specific aspects of EGF receptor signaling and might become valuable as anticancer agents. Intracellular expression of the aptamer encoding gene construct KDI1 or introduction of bacterially expressed KDI1 via a protein transduction domain into EGFR-expressing cells results in KDI1.EGF receptor complex formation, a slower proliferation, and reduced soft agar colony formation. Aptamer KDI1 did not summarily block the EGF receptor tyrosine kinase activity but selectively interfered with the EGF-induced phosphorylation of the tyrosine residues 845, 1068, and 1148 as well as the phosphorylation of tyrosine 317 of p46 Shc. EGF-induced phosphorylation of Stat3 at tyrosine 705 and Stat3-dependent transactivation were also impaired. Transduction of a short synthetic peptide aptamer sequence not embedded into the scaffold protein resulted in the same impairment of EGF-induced Stat3 activation.