Chang Deok Kim

Can Platelet-rich Plasma Be Used for Skin Rejuvenation? Evaluation of Effects of Platelet-rich Plasma on Human Dermal Fibroblast

Background Autologous platelet-rich plasma has attracted attention in various medical fields recently, including orthopedic, plastic, and dental surgeries and dermatology for its wound healing ability. Further, it has been used clinically in mesotherapy for skin rejuvenation. Objective In this study, the effects of activated platelet-rich plasma (aPRP) and activated platelet-poor plasma (aPPP) have been investigated on the remodelling of the extracellular matrix, a process that requires activation of dermal fibroblasts, which is essential for rejuvenation of aged skin. Methods Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared using a double-spin method and then activated with thrombin and calcium chloride. The proliferative effects of aPRP and aPPP were measured by [3H]thymidine incorporation assay, and their effects on matrix protein synthesis were assessed by quantifying levels of procollagen type I carboxy-terminal peptide (PIP) by enzyme-linked immunosorbent assay (ELISA). The production of collagen and matrix metalloproteinases (MMP) was studied by Western blotting and reverse transcriptase-polymerase chain reaction. Results Platelet numbers in PRP increased to 9.4-fold over baseline values. aPRP and aPPP both stimulated cell proliferation, with peak proliferation occurring in cells grown in 5% aPRP. Levels of PIP were highest in cells grown in the presence of 5% aPRP. Additionally, aPRP and aPPP increased the expression of type I collagen, MMP-1 protein, and mRNA in human dermal fibroblasts. Conclusion aPRP and aPPP promote tissue remodelling in aged skin and may be used as adjuvant treatment to lasers for skin rejuvenation in cosmetic dermatology.

Autophagy Negatively Regulates Keratinocyte Inflammatory Responses via Scaffolding Protein p62/SQSTM1

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern‐recognition receptors expressed in keratinocytes. We found that keratinocyte Toll‐like receptors (TLRs) 2 and 4 and Dectin‐1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL‐37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin‐1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL‐37. In addition, TLR2, Dectin‐1 and, to an extent, TLR4 are essential for the MU‐mediated expression of CXCL8, CCL2 and LL‐37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin‐1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL‐37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin‐1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

Propionibacterium acnes Activates the NLRP3 Inflammasome in Human Sebocytes

Propionibacterium acne and sebaceous glands are considered to have an important role in the development of acne. Although information regarding the activation of innate immunity by P. acnes in the sebaceous gland is limited, different P. acnes phylotypes and a higher prevalence of follicular P. acnes macrocolonies/biofilms in sebaceous follicles of skin biopsies from acne compared with control skin and occasionally single P. acnes clusters in single sebaceous glands have been detected. In this study, we investigated whether P. acnes activates the inflammasome in human sebaceous glands in vivo and in vitro. We found that IL-1β expression was upregulated in sebaceous glands of acne lesions. After stimulation of human sebocytes with P. acnes, the activation of caspase-1 and secretion of IL-1β were enhanced significantly. Moreover, knocking down the expression of NLRP3 abolished P. acnes-induced IL-1β production in sebocytes. The activation of the NLRP3 inflammasome by P. acnes was dependent on protease activity and reactive oxygen species generation. Finally, we found that NALP3-deficient mice display an impaired inflammatory response to P. acnes. These results suggest that human sebocytes are important immunocompetent cells that induce the NLRP3 inflammasome, and that P. acnes-induced IL-1β activation in sebaceous glands may have a role in combating skin infections and in acne pathogenesis.

Differential Gene Expression Profiles of Radioresistant Non–Small-Cell Lung Cancer Cell Lines Established by Fractionated Irradiation: Tumor Protein p53-Inducible Protein 3 Confers Sensitivity to Ionizing Radiation

PURPOSE Despite the widespread use of radiotherapy as a local and regional modality for the treatment of cancer, some non-small-cell lung cancers commonly develop resistance to radiation. We thus sought to clarify the molecular mechanisms underlying resistance to radiation. METHODS AND MATERIALS We established the radioresistant cell line H460R from radiosensitive parental H460 cells. To identify the radioresistance-related genes, we performed microarray analysis and selected several candidate genes. RESULTS Clonogenic and MTT assays showed that H460R was 10-fold more resistant to radiation than H460. Microarray analysis indicated that the expression levels of 1,463 genes were altered more than 1.5-fold in H460R compared with parental H460. To evaluate the putative functional role, we selected one interesting gene tumor protein p53-inducible protein 3 (TP53I3), because that this gene was significantly downregulated in radioresistant H460R cells and that it was predicted to link p53-dependent cell death signaling. Interestingly, messenger ribonucleic acid expression of TP53I3 differed in X-ray-irradiated H460 and H460R cells, and overexpression of TP53I3 significantly affected the cellular radiosensitivity of H460R cells. CONCLUSIONS These results show that H460R may be useful in searching for candidate genes that are responsible for radioresistance and elucidating the molecular mechanism of radioresistance.

Pitx2, a β-catenin-regulated transcription factor, regulates the differentiation of outer root sheath cells cultured in vitro

BACKGROUND Beta-catenin exerts its crucial role in hair follicle development and hair growth cycle. Although the importance of Wnt/beta-catenin is well recognized, the downstream effectors of beta-catenin have not been clearly elucidated yet. OBJECTIVE The aim of this study is to identify the beta-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells. METHODS We transduced ORS cells with adenovirus harboring the expression cassette for constitutive active form of beta-catenin, then performed cDNA microarray. RESULTS Overexpression of beta-catenin led to the upregulation of hair cell differentiation markers such as keratin 16 and 17. In addition, the expression of Pitx2, a bicoid-type homeodomain transcription factor, was also increased by overexpression of beta-catenin in ORS cells cultured in vitro. To investigate the potential role of Pitx2, we made the recombinant adenovirus expressing Pitx2, then transduced into the cultured ORS cells. Interestingly, Pitx2 induced the expression of keratin 16 and 17, indicating that Pitx2 activates ORS cells towards the follicular differentiation pathway preferentially. CONCLUSION Our results implicate the potential importance of Pitx2 as a beta-catenin downstream modulator in hair growth control.

Expression and Functional Role of Sox9 in Human Epidermal Keratinocytes

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.

Regulation of lipid production by acetylcholine signalling in human sebaceous glands

BACKGROUND The extraneuronal cholinergic system has been implicated in numerous functions in the skin, such as terminal differentiation, barrier formation, sweat secretion and the microcirculation. However, the evidence for cholinergic signalling in sebaceous glands is lacking, and its role needs to be clarified. OBJECTIVE We investigated the role of acetylcholine signalling in sebaceous glands using human sebocytes and a clinical study using botulinum toxin. METHODS Immunohistochemistry and immunocytofluorescence were performed to evaluate cholinergic receptor levels in sebaceous glands. Lipid levels were assessed by Oil Red O staining and signalling pathways by Western blotting. To evaluate the clinical relevance, we also assessed the effect of botulinum toxin on sebum production in healthy volunteers. RESULTS We demonstrated that human skin sebaceous glands in vivo and sebocytes in vitro express nicotinic acetylcholine receptor α7 (nAchRα7), and that acetylcholine increased lipid synthesis in a dose-dependent manner. When sebocytes were incubated with α-bungarotoxin, a competitive nAchR antagonist, acetylcholine failed to up-regulate lipid synthesis. Twenty healthy volunteers were enrolled in a double-blind, placebo-controlled, split-face study. A marked decrease in sebum production on the botulinum-treated side was found in volunteers with oily skin. CONCLUSION These results provide evidence that acetylcholine signalling plays a significant role in human sebaceous gland biology and identify acetylcholine signalling as a promising target in the clinical management of disorders in which sebum production is increased, such as acne vulgaris.

Apurinic/Apyrimidinic Endonuclease 1 Is a Key Modulator of Keratinocyte Inflammatory Responses

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1α and NF-κB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-α, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Identification of calcium-inducible genes in primary keratinocytes using suppression-subtractive hybridization.

Abstract:  Terminal differentiation in epidermal keratinocytes involves major biochemical changes including the expression of many new differentiation‐specific genes. To further understand this process, we performed suppression‐subtractive hybridization of keratinocytes cultured under high‐calcium condition, known to induce differentiation in vitro. We randomly isolated 300 clones representing 90 different genes. By reverse Northern blot analyses, 20 different genes were found to be overexpressed, of which 13 were confirmed as differentially expressed genes during keratinocyte differentiation by Northern blot analysis. Of those, five genes, transglutaminase 1, keratin 6, interleukin‐1 receptor antagonist, kallikrein 7, and heat shock protein 27, are known to be up‐regulated during epidermal differentiation. Six genes, ferritin‐L chain, ribosomal protein S6, tumor‐associated calcium signal transducer 2, neuroendocrine secretory protein 55, phosphoserine aminotransferase, and neutrophil gelatinase‐associated lipocalin, heretofore were not known to be up‐regulated during keratinocyte differentiation. We also identified two novel genes. One of these maps to chromosome 1q21 of the epidermal differentiation complex, and its expression level was strongly increased in differentiating keratinocytes. These differentially expressed genes may provide significant opportunities for further understanding of the epidermal keratinocyte differentiation.