Keratinocyte-conditioned Medium Research Articles

The ginsenoside Rg2 downregulates MMP-1 expression in keratinocyte (HaCaT)-conditioned medium-treated human fibroblasts (Hs68)

Keratinocytes exposed to UVB induce the production of cytokines, which activate fibroblasts and increase the expression of matrix metalloproteinases (MMPs). The increased expression of MMPs leads to connective tissue damage and wrinkle formation, resulting in skin aging. In this study, we used human dermal fibroblasts cultured in UVB-irradiated keratinocyte-conditioned medium (UV CM) to investigate the potential anti-aging effects of the ginsenoside Rg2 on skin. The inhibitory effect of Rg2 on the MMP-1 gene and protein was determined by real-time PCR and ELISA. We also examined the expression levels of proteins in the mitogen-activated protein kinase (MAPK) signaling pathway using western blotting, to elucidate the underlying mechanism of the inhibitory effect of Rg2. Rg2 inhibited MMP-1 mRNA and protein expression in a concentration-dependent manner. We found that Rg2 inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not that of p38. Therefore, our results suggest that Rg2 is a potential material for the prevention and treatment of photoaging.

Visualizing Activated Myofibroblasts Resulting from Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that exhibit two main characteristics which define stem cells: self-renewal and differentiation. MSCs can migrate to sites of injury, inflammation, and tumor. Moreover, MSCs undergo myofibroblast-like differentiation, including increased production of α-SMA in response to transforming growth factor-β (TGF-β), a growth factor commonly secreted by tumor cells to evade immune surveillance. Based on our previous findings, hMSCs become activated and resemble carcinoma-associated myofibroblasts upon prolonged exposure to a conditioned medium from MDAMB231 human breast cancer cells. In this section, we show using immunofluorescence that keratinocyte-conditioned medium (KCM) induces differentiation of MSCs to resemble dermal myofibroblast-like cells with punctate vinculin staining and F-actin filaments.

RAB3A Regulates Melanin Exocytosis and Transfer Induced by Keratinocyte-Conditioned Medium

Skin pigmentation is imparted by melanin and is crucial for photoprotection against UVR. Melanin is synthesized and packaged into melanosomes within melanocytes and is then transferred to keratinocytes (KCs). Although the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain unclear. Previously, our group proposed that coupled exocytosis/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for RAB11B and the exocyst tethering complex in this process. In this study, we show that soluble factors present in KC-conditioned medium stimulate melanin exocytosis from melanocytes and transfer to KCs. Moreover, we found that these factors are released by differentiated KCs but not by basal layer KCs. Furthermore, we found that RAB3A regulates melanin exocytosis and transfer stimulated by KC-conditioned medium. Indeed, KC-conditioned medium enhances the recruitment of RAB3A to melanosomes in melanocyte dendrites. Therefore, our results suggest the existence of two distinct routes of melanin exocytosis: a basal route controlled by RAB11B and a RAB3A-dependent route, stimulated by KC-conditioned medium. Thus, this study provides evidence that soluble factors released by differentiated KCs control skin pigmentation by promoting the accumulation of RAB3A-positive melanosomes in melanocyte dendrites and their release and subsequent transfer to KCs.

Role of the Skin Microenvironment in Melanomagenesis: Epidermal Keratinocytes and Dermal Fibroblasts Promote BRAF Oncogene-Induced Senescence Escape in Melanocytes.

Simple SummaryMelanoma is a deadly skin cancer caused by the uncontrolled proliferation of melanocytes, a population of specialized cells that produce the skin pigment melanin. An aberrant proliferation of melanocytes is common, manifesting as new moles, and these lesions often remain benign. Only a small fraction of these aberrant melanocytes transition to melanoma (i.e., melanomagenesis). The factors that drive this transition are not fully understood. Recent studies have suggested that other cells—specifically, keratinocytes that make up the upper skin layers and fibroblasts, which are non-specialized cells within the deeper layers of the skin—also contribute to melanomagenesis. Here, employing microscale models that mimicked the skin microenvironment, we investigated the effect of crosstalk between melanocytes as well as keratinocytes and fibroblasts on melanomagenesis. Our findings show that keratinocyte- and fibroblast-derived factors can inhibit the mechanisms that prevent an uncontrolled melanocyte proliferation and contribute to melanomagenesis. Thus, targeting skin microenvironment-derived factors is a potential strategy to prevent melanomagenesis.BRAFV600E is the most common mutation driver in melanoma. This mutation is known to cause a brief burst of proliferation followed by growth arrest and senescence, which prevent an uncontrolled cell proliferation. This phenomenon is known as oncogene-induced senescence (OIS) and OIS escape is thought to lead to melanomagenesis. Much attention has been focused on the melanocyte-intrinsic mechanisms that contribute to senescence escape. Additional genetic events such as the loss of tumor suppressor PTEN and/or epigenetic changes that contribute to senescence escape have been described. However, the role of the skin microenvironment—specifically, the role of epidermal keratinocytes—on melanomagenesis is not fully understood. In this study, we employ a microfluidic platform to study the interaction between melanocytes expressing the BRAFV600E mutation as well as keratinocytes and dermal fibroblasts. We demonstrate that keratinocytes suppress senescence-related genes and promote the proliferation of transformed melanocytes. We also show that a keratinocyte-conditioned medium can alter the secretion of both pro- and anti-tumorigenic factors by transformed melanocytes. In addition, we show that melanocytes and keratinocytes from donors of white European and black African ancestry display different crosstalks; i.e., white keratinocytes appear to promote a more pro-tumorigenic phenotype compared with black keratinocytes. These data suggest that keratinocytes exert their influence on melanomagenesis both by suppressing senescence-related genes in melanocytes and by affecting the balance of the melanocyte-secreted factors that favor tumorigenesis.

Tranexamic acid inhibits melanogenesis partially via stimulation of TGF-β1 expression in human epidermal keratinocytes.

Oral tranexamic acid (TA) has been an effective treatment for melasma with unclear mechanism. The present study aimed to demonstrate the effect of TA on melanogenesis via regulation of TGF-β1 expression in keratinocytes. We firstly determined the expression level of TGF-β1 in TA-treated keratinocyte-conditioned medium (KCM). Then, the mRNA and protein levels of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1) of human epidermal melanocytes (NHEMs) in the presence of TA-treated KCM were evaluated via RT-PCR and western blot analysis. Moreover, melanin content and tyrosinase activity were quantified. TGF-β1 gene was knocked down by small interfering RNA (siRNA) in keratinocytes. The mRNA and protein levels of TGF-β1 in keratinocytes were significantly increased after TA treatment. Melanin contents, tyrosinase activity, protein and mRNA levels of TYR, MITF and TRP-1 were downregulated in NHEMs in the presence of TA-treated KCM. Knockdown of TGF-β1 in keratinocytes could attenuate the inhibitory effect of TA-treated KCM on melanogenesis. TA could stimulate TGF-β1 expression in keratinocytes, which further inhibits melanogenesis through the paracrine signalling.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

Regenerative potential of partially differentiated mesenchymal stromal cells in a mouse model of a full-thickness skin wound.

Mesenchymal stromal cells (MSCs, known as mesenchymal stem cells) are considered to be a promising therapeutic tool for many diseases. But it is still unclear which cells are more efficient and safe for wound healing and tissue regeneration for clinical applications: undifferentiated, partially differentiated stem cells or differentiated cells. In this study, we modified MSCs with keratinocyte-conditioned medium (KCM) and examined MSCs, partially differentiated MSCs (PMSCs) and differentiated cell migration, accumulation in the wounded area as well as cell regenerative efficiency in a full-thickness skin wound model. In addition to that, the impact of intradermal and intravenous cell delivery methods of wound healing was evaluated. C57BL/6J mouse compact bone MSCs were treated with a KCM for 14 days. Flow cytometry analysis showed the appearance of keratinocyte surface markers which were absent in MSCs, whereas the specific markers for MSCs were lost. Cells were injected either intravenously or intradermally in C57BL/6J mice. Wound closure, cell migration and accumulation in the wounded area were further analysed. Wound healing was assessed by the rate of wound closure and by histological evaluation. Cells were monitored using optical imaging. We demonstrated that PMSCs showed morphology similar to keratinocyte cells, had enhanced migration and increased survival at the site of injury. PMSCs had a beneficial effect on wound healing and tissue regeneration. This effect was reinforced when these cells were injected intravenously. Due to their partial differentiation status, we assume that PMSCs can differentiate more rapidly into epidermal cell lineages thus causing faster and qualitatively improved wound healing.

Scopoletin downregulates MMP‑1 expression in human fibroblasts via inhibition of p38 phosphorylation.

Irradiation of keratinocytes by ultraviolet B induces cytokine production, which in turn activates fibroblasts to produce cytokines and increase matrix metallopeptidase (MMP)‑1 protein expression. The present study investigated the effect and potential mechanisms of scopoletin on the regulation of MMP‑1 expression in fibroblasts. Scopoletin was isolated from Artemisiacapillaris crude extract. Treatment of fibroblasts with scopoletin resulted in a decrease in the protein expression of MMP‑1 following stimulation with human keratinocyte (HaCaT) conditioned medium. To further explore the mechanism underlying this effect, the expression levels of proteins in the mitogen‑activated protein kinase (MAPK) and nuclear factor‑κB (NF‑κB) signaling pathways were evaluated via western blot analysis. The mRNA expression levels of interleukin (IL)‑1α and tumor necrosis factor (TNF) α were evaluated via reverse transcription‑quantitative polymerase chain reaction. The effect of scopoletin on cell viability was assessed with the MTT assay. The results demonstrated that scopoletin treatment markedly decreased MMP‑1, IL‑1α and TNFα mRNA expression in fibroblasts stimulated with HaCaT conditioned medium (40mJ/cm2), without any apparent cell cytotoxicity, and in a dose‑dependent manner. In addition, western blot analysis demonstrated that scopoletin reduced the phosphorylation of p38 MAPK in fibroblasts. In summary, the present study demonstrated that scopoletin inhibited MMP‑1 and proinflammatory cytokine expression by inhibiting p38 MAPK phosphorylation. These findings suggest that scopoletin may have potential as a therapeutic agent to prevent and treat photoaging of the skin.

Effect of TGF\u03b21, TGF\u03b23 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin’s pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor β family (TGFβ) expression showed significantly higher levels of Tgfβ3 transcript between nude and Balb/c mice but no differences were detected for Tgfβ1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFβ3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFβ3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Opioids and opioid receptors orchestrate wound repair

We have previously shown that topical opioids including morphine and its congeners promote healing of full thickness ischemic wounds in rats. We examined the contribution of mu opioid receptor (MOPr)-mediated healing of full thickness ischemic wounds using MOPr and delta or kappa opioid receptor knockout (KO) mice. Wound closure in the early (day 5) as well as later phases was delayed in topical morphine or PBS-treated MOPr-KO mice compared with reciprocal treatments of wounds in wild-type (WT) mice. MOPr expression was significantly upregulated at 30min in the wound margins and colocalized with wound margins and vasculature in the epidermal and dermal layers of the skin. We next examined whether neuropeptide expression was involved in the mechanism of MOPr-mediated wound closure. Substance P (SP) and calcitonin gene-related peptide immunoreactivity (ir) was significantly increased in the skin of MOPr-KO mice as compared with WT mice. Neuropeptide-ir was increased significantly in PBS-treated wounds of MOPr and WT mice, but morphine treatment reduced neuropeptide immunoreactivity in both as compared with PBS. Wounding of keratinocytes led to the release of opioid peptide beta-endorphin (β-END) in conditioned medium, which stimulated the proliferation of endothelial cells. MOPr-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, CTOP) and nonselective OPr antagonist naloxone-inhibited endothelial proliferation induced by wounded keratinocyte-conditioned medium. In addition, accelerated wound area closure invitro by morphine was suppressed by methylnaltrexone, a nonselective OPr antagonist with high affinity for MOPr. Morphine and its congeners stimulated the proliferation of endothelial cells from WT mice but not those from MOPr-KO mice. Furthermore, morphine-induced mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation in endothelial cells was significantly decreased in MOPr-KO mice as compared with WT mice. Collectively, these data suggest that MOPr plays a critical role in the proliferation phase with the formation of granulation tissue during wound healing.

Activation and Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells and exhibit two main characteristics that define stem cells: self-renewal and differentiation. MSCs can migrate to sites of injury, inflammation, and tumor. Moreover, MSCs undergo myofibroblast like differentiation, including increased production of α-SMA in response to transforming growth factor-β (TGF-β), a growth factor commonly secreted by tumor cells to evade immune surveillance. Based on our previous finding hMSCs become activated and resemble carcinoma-associated myofibroblasts upon prolonged exposure to conditioned medium from MDAMB231 human breast cancer cells. Here, we show that keratinocyte conditioned medium (KCM) induces differentiation of MSCs to resemble dermal myofibroblast like cells using immunofluorescence techniques demonstrating punctate vinculin staining, and F-actin filaments.

Keratinocyte-Releasable Factors Stimulate the Expression of Granulocyte Colony-Stimulating Factor in Human Dermal Fibroblasts.

Interaction between keratinocytes and fibroblasts plays a critical role in maintaining skin integrity under both normal and pathological conditions. We have previously demonstrated that keratinocyte-releasable factors influence the expression of key extracellular matrix components, such as collagen and matrix metalloproteinases in dermal fibroblasts. In this study, we utilized DNA microarray analysis to examine the effects of keratinocyte-releasable factors on the expression of several cytokines in human dermal fibroblasts. The results revealed significantly higher granulocyte colony-stimulating factor (G-CSF) expression in fibroblasts co-cultured with keratinocytes relative to mono-cultured cells, which was verified by RT-PCR and western blot. G-CSF is an important hematopoietic factor also thought to play a beneficial role in wound healing through stimulating keratinocyte proliferation. To partially characterize the keratinocyte-releasable factors responsible for stimulating G-CSF production, keratinocyte-conditioned medium (KCM) was subjected to thermal treatment and ammonium sulfate precipitation before treating fibroblasts. The results showed that keratinocyte-releasable G-CSF-stimulating factors remain stable at 56°C and upon 50% ammonium sulfate precipitation. Knowing that keratinocytes release IL-1, which stimulates G-CSF expression in various immune cells, several experiments were conducted to ask whether this might also be the case for fibroblasts. The results showed that the addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression, indicating the role of additional keratinocyte-releasable factors. These findings underline the importance of cross-talk between keratinocytes and fibroblasts, suggesting that communication between these cells in vivo modulates the production of cytokines required for cutaneous wound healing and maintenance. J. Cell. Biochem. 118: 308-317, 2017. © 2016 Wiley Periodicals, Inc.

IL-22 capacitates dermal fibroblast responses to TNF in scleroderma

ObjectivesInterleukin (IL) 22 mRNA in systemic sclerosis (SSc) skin and Th22 cells in SSc peripheral blood are increased, but the role of IL-22 in fibrosis development remains poorly understood.MethodsBiopsies were...

Expression profiling of human melanocytes in response to UV-B irradiation.

A comprehensive gene expression analysis of human melanocytes was performed assessing the transcriptional profile of dark melanocytes (DM) and light melanocytes (LM) at basal conditions and after UV-B irradiation at different time points (6, 12 and 24 h), and in culture with different keratinocyte-conditioned media (KCM + and KCM −). The data, previously published in [1], have been deposited in NCBI's Gene Expression Omnibus (GEO accession number: GSE70280).

Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium.

IntroductionBone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation.MethodsBMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses.ResultsThe ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses.ConclusionsThis study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.

Proteolytic and Non-proteolytic Activation of Keratinocyte-Derived Latent TGF-β1 Induces Fibroblast Differentiation in a Wound-Healing Model Using Rat Skin

Transforming growth factor-β1 (TGF-β1) reportedly causes the differentiation of fibroblasts to myofibroblasts during wound healing. We investigated the mechanism underlying the activation of latent TGF-β1 released by keratinocytes in efforts to identify promising pharmacological approaches for the prevention of hypertrophic scar formation. A three-dimensional collagen gel matrix culture was prepared using rat keratinocytes and dermal fibroblasts. Stratified keratinocytes promoted the TGF receptor–dependent increase in α-smooth muscle actin (α-SMA) immunostaining and mRNA levels in fibroblasts. Latent TGF-β1 was found to be localized suprabasally and secreted. α-SMA expression was inhibited by an anti-αv-integrin antibody and a matrix metalloproteinase (MMP) inhibitor, GM6001. In a two-dimensional fibroblast culture, α-SMA expression depended on the production of endogenous TGF-β1 and required αv-integrin or MMP for the response to recombinant latent TGF-β1. In keratinocyte-conditioned medium, MMP-dependent latent TGF-β1 secretion was detected. Applying this medium to the fibroblast culture enhanced α-SMA production. This effect was decreased by GM6001, the anti-αv-integrin antibody, or the preabsorption of latent TGF-β1. These results indicate that keratinocytes secrete latent TGF-β1, which is liberated to fibroblasts over distance and is activated to produce α-SMA with the aid of a positive-feedback loop. MMP inhibition was effective for targeting both keratinocytes and fibroblasts in this model. [Supplementary Figure: available only at http://dx.doi.org/10.1254/jphs.13209FP]

Transdifferentiation of Adipose-Derived Stem Cells into Keratinocyte-Like Cells: Engineering a Stratified Epidermis

Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC) have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS). After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC); these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca2+ concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca2+. Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a readily available and accessible source of keratinocytes, particularly for severe wounds encompassing large surface areas of the body and requiring prompt epithelialization.

Progression of genotype-specific oral cancer leads to senescence of cancer-associated fibroblasts and is mediated by oxidative stress and TGF-β

Keratinocyte senescence acts as a barrier to tumor progression but appears to be lost in late pre-malignancy to yield genetically unstable oral squamous cell carcinomas (GU-OSCC); a subset of OSCC possessing wild-type p53 and are genetically stable (GS-OSCC). In this study, fibroblasts from GU-OSCC were senescent relative to fibroblasts from GS-OSCC, epithelial dysplastic tissues or normal oral mucosa, as demonstrated by increased senescence-associated β-galactosidase (SA β-Gal) activity and overexpression of p16(INK4A). Keratinocytes from GU-OSCC produced high levels of reactive oxygen species (ROS) and this was associated with an increase in the production of transforming growth factor-β1 (TGF-β1) and TGF-β2 in stromal fibroblasts. Treatment of normal fibroblasts with keratinocyte conditioned media (CM) from GU-OSCC, but not GS-OSCC or dysplastic keratinocytes with dysfunctional p53, induced fibroblast senescence. This phenomenon was inhibited by antioxidants and anti-TGF-β antibodies. Fibroblast activation by TGF-β1 preceded cellular senescence and was associated with increased ROS levels; antioxidants inhibited this reaction. Senescent fibroblasts derived from GU-OSCC or normal fibroblasts treated with CM from GU-OSCC or hydrogen peroxide, but not non-senescent fibroblasts derived from GS-OSCC, promoted invasion of keratinocytes in vitro. Epithelial invasion was stimulated by fibroblast activation and amplified further by fibroblast senescence. The data demonstrate that malignant keratinocytes from GU-OSCC, but not their pre-malignant counterparts, produce high levels of ROS, which, in turn, increase TGF-β1 expression and induce fibroblast activation and senescence in a p5-independent manner. Fibroblasts from GU-OSCC were particularly susceptible to oxidative DNA damage because of high levels of ROS production, downregulation of antioxidant genes and upregulation of pro-oxidant genes. The results demonstrate the functional diversity of cancer-associated fibroblasts and show that malignant keratinocytes from GU-OSCC reinforce their malignant behavior by inducing fibroblast activation and senescence through ROS and TGF-β-dependent mechanisms.

Antiphotoaging Effects of Light-Emitting Diode Irradiation on Narrow-Band Ultraviolet B–Exposed Cultured Human Skin Cells

Antiaging effects of light-emitting diodes (LEDs) have been clinically demonstrated using one or two wavelengths. The mechanism is unclear. To examine direct and indirect photobiomodulation effects of LEDs on narrow-band ultraviolet B (NB-UVB)-induced photoaging using seven different wavelengths alone or in combination. Four LED wavelengths were chosen based on type I collagen and metalloproteinase (MMP)-1 expression. NB-UVB-irradiated fibroblasts or keratinocytes were irradiated using these four wavelengths. The expression of collagen and MMP-1 in fibroblasts with or without conditioned medium from LED-irradiated keratinocytes and the expression of proinflammatory cytokines in the LED-irradiated keratinocytes were examined. Irradiation with four wavelengths (630, 660, 830, and 850nm) significantly increased the number of viable fibroblasts. These four wavelengths also increased type I collagen expression, particularly four combinations (630/830, 660/850, 630/850, and 660/830nm). The fibroblasts cultured with the keratinocyte conditioned medium, particularly with a combination of 630/850 or 660/830nm, increased collagen levels. Low tumor necrosis factor alpha (TNF-α) and high transforming growth factor beta 1 (TGF-β1) expression was detected in the LED-irradiated keratinocytes. The combination of 630/850- or 660/830-nm irradiation led to better direct and indirect antiphotoaging outcomes than single LED wavelengths in NB-UVB-irradiated cultured normal human skin cells.

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.