Effects of Xanthium stramarium and Psoralea corylifolia Extracts Combined with UVA1 Irradiation on the Cell Proliferation and TGF-β1 Expression of Keloid Fibroblasts

Background

Xanthium stramarium (XAS) and Psoralea corylifolia (PSC), phototoxic oriental medicinal plants, has been used in traditional medicines in Asian countries.

Objective

The effects of highly purified XAS or PSC extract combined with ultraviolet A1 (UVA1) irradiation on cell proliferation and transforming growth factor-beta1 (TGF-β1) expression of the keloid fibroblast were being investigated to define potential therapeutic uses for keloid treatments.

Methods

The keloid fibroblasts were treated with XAS or PSC alone or in the combination with UVA1 irradiation. The cell viability, apoptosis, and expression of TGF-β1 and collagen I were investigated.

Results

XAS and PSC in combination with UVA1 irradiation suppressed cell proliferation and induced apoptosis of keloid fibroblasts. Furthermore, the XAS and PSC in combination with UVA1 irradiation inhibited TGF-β1 expression and collagen synthesis in keloid fibroblasts.

Conclusion

These findings may open up the possibility of clinically used XAS or PSC in combination with UVA1 irradiation for keloid treatments.     

Expression of Toll-like Receptor 2 in Cultured Human Keratinocytes: The Effect of Bacterial Antigens, Cytokines and Calcium Concentration

Background

Toll-like receptors (TLRs) are expressed by human epidermal keratinocytes and are involved in immune responses.

Objective

The goal of this was to investigate the expression of TLR2 in response to bacterial antigens, cytokines, and different calcium concentrations.

Methods

The expression of TLR2 was assessed after stimulation by lipoteichoic acid (LTA) and streptolysin O (SLO). In addition, TLR2 expression was evaluated after treatment with IFN-γ and TNF-α, and different concentrations of calcium. The expression levels of TLR2 mRNA and protein were studied using RT-PCR and Western blot analysis.

Results

Cultured human epidermal keratinocytes constitutively expressed TLR2 and the expression was stimulated by LTA and SLO; in addition, IFN-γ and TNF-α upregulated TLR2 expression. However, the changes in TLR2 expression associated with the calcium concentrations were insignificant.

Conclusion

TLR2 expression increased with the concentration and duration of bacterial pathogens and this increase was amplified by several cytokines, from activated keratinocytes and other cells.     

Interleukin-17 and Interleukin-22 Induced Proinflammatory Cytokine Production in Keratinocytes via Inhibitor of Nuclear Factor ��B Kinase-�� Expression

Background

The pathogenesis of psoriasis may involve the interleukin (IL)-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22, which mediates dermal inflammation and acanthosis.

Objective

As inhibitor of nuclear factor κB kinase-α (IKKα) has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKα.

Methods

We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching to the high concentration (2.8 mM) media with IL-17 or IL-22, then compared the IKKα expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies.

Results

Elevated calcium concentration induced IKKα expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKα in HaCaT cells and reconstituted human epidermis. IKKα induction was also noted, following the injection of IL-17 and IL-22 into mice ears.

Conclusion

Although the induction of IKKα was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to the inflammation occurring via the induction of IKKα from keratinocytes or skin layers.     

Stimulation of the Extracellular Matrix Production in Dermal Fibroblasts by Velvet Antler Extract

Background

Fibroblasts produce many components of the extracellular matrix (ECM) and so they contribute to the maintenance of connective tissue integrity.

Objective

The aim of this study is to evaluate the effect of velvet antler extract (VAE) on the ECM production of dermal fibroblasts cultured in vitro.

Methods

Primary cultured human dermal fibroblasts were treated with VAE, and then the ECM production was determined by RT-PCR, ELISA and Western blot analysis. Furthermore, the change of gene expression according to VAE treatment was evaluated by cDNA microarray.

Results

VAE accelerated the growth of fibroblasts in a dose-dependent manner. VAE increased the production of several ECM components, including type 1 collagen, fibronectin and elastin. In line with these results, the phosphorylations of p42/44 ERK and p38 mitogen-activated protein kinase were markedly increased by VAE, suggesting that the enhancement of ECM production may be linked to the activation of intracellular signaling cascades. VAE also significantly increased cell migration on an in vitro scratch wound test. In cDNA microarray, many genes related with connective tissue integrity were identified to be up-regulated by VAE.

Conclusion

These results suggest that VAE has a potential to stimulate ECM production, and VAE may be applicable for maintaining the skin''s texture.     

Expression Patterns of Gli-1, Pleckstrin Homology-Like Domain,Family A,Member 1, Transforming Growth Factor-β1/β2, and p63 in Sebaceous and Follicular Tumors

Background

Certain epidermal appendage tumors, including hyperplasias (hamartomas), adenomas, benign epitheliomas, primordial epitheliomas, and malignant tumors, can exhibit any stage of differentiation. Several molecules associated with tumorigenesis, such as Gli-1, pleckstrin homology-like domain, family A, member 1 (PHLDA-1), transforming growth factor (TGF)-β1, TGF-β2, and p63, are associated with tumor grade and aggressive behavior in follicular and sebaceous tumors in ways that are not well understood.

Objective

The aim of this study was to elucidate the expression of Gli-1, PHLDA-1, TGF-β1/β2, and p63 in benign and malignant tumors of the hair and sebaceous glands and to determine their importance in the degree of tumor differentiation.

Methods

Immunohistochemistry was performed in follicular and sebaceous tumors using antibodies against Gli-1 (sebaceous tumor marker), PHLDA-1 (hair follicle outer root sheath [ORS] cell marker), p63, TGF-β1, and TGF-β2.

Results

Gli-1 was expressed in basaloid cells, sebocytes, and sebaceous carcinoma cells, and expression levels decreased as differentiation progressed. PHLDA-1 was expressed in ORS cells and some follicular tumor cells. Expression of p63 was observed in the nuclei of the outermost basaloid cells (seboblasts), poorly differentiated sebaceous carcinoma cells, and tumor cells toward the direction of the hair. Remarkably, TGF-β1 was expressed exclusively in the nuclei of benign and malignant follicular (hair) tumors, but not in sebaceous tumors, at levels that correlated with the degree of differentiation.

Conclusion

We propose that p63 and/or TGF-β1 are useful for predicting the degree of differentiation and malignant potential of sebaceous and follicular tumors and for distinguishing trichilemmal carcinoma from sebaceous carcinoma.     

Epidermal growth factor-induced matrix metalloproteinase-1 expression is negatively regulated by p38 MAPK in human skin fibroblasts

Background

Many extracellular stimuli, including epidermal growth factor (EGF), are known to induce MMP-1 expression. Recently, several reports have shown that ERK activity plays an important role in EGF-induced MMP-1 expression. However, EGF is also known to activate many signaling pathways in addition to the ERK pathway, but the roles of these pathways during the induction of MMP-1 by EGF are unclear.

Objective

We investigated the role of JNK, p38 MAPK, and PI3K/Akt pathways in EGF-induced MMP-1 expression in human skin fibroblasts. Then, we further explored the inhibitory effect of p38 MAPK pathway on EGF-induced MMP-1 expression and studied the molecular mechanisms involved in the processes.

Methods

Human skin fibroblasts were pretreated with various chemical inhibitors or small interfering RNA (siRNA) at the indicated concentrations and then treated with EGF, TNF-alpha, or IL-1beta for the indicated times. Protein and mRNA levels of various target molecules were assessed by Western blotting and quantitative real-time PCR, respectively.

Results

We found that EGF-induced MMP-1 expression was positively regulated by JNK as well as ERK but negatively regulated by p38 MAPK in human skin fibroblasts. On the other hand, the PI3K/Akt pathway did not significantly affect MMP-1 induction by EGF. Then we found that the inhibition of p38 MAPK pathway specifically increased the MMP-1 expression stimulated by EGF but not by TNF-alpha or IL-1beta, indicating that the effect of p38 MAPK on MMP-1 expression may be stimulus-type specific in human skin fibroblasts. In addition, the inhibitory effect of p38 MAPK on EGF-induced MMP-1 expression was shown to be mainly mediated by p38-alpha MAPK. Our further studies showed that the inhibition of p38 MAPK but not PI3K specifically increased EGF-induced ERK and JNK activations, and that the augmentation of EGF-induced MMP-1 expression by p38 MAPK inhibition was significantly attenuated by inhibiting the activities of ERK and/or JNK.

Conclusions

Our results indicate that EGF-induced MMP-1 expression is differentially regulated by the JNK, p38 MAPK, and PI3K/Akt pathways, and suggest that p38 MAPK negatively regulates EGF-induced MMP-1 expression by suppressing the activations of ERK and JNK.     

Effects of Human Adipose-derived Stem Cells on Cutaneous Wound Healing in Nude Mice

Background

Despite numerous treatments available for deteriorated cutaneous wound healing such as a diabetic foot, there is still the need for more effective therapy. Adipose-derived stem cells (ASCs) are mesenchymal stem cells, which are self-renewing and multipotent. Mesenchymal stem cells have the potential for tissue repair and regeneration.

Objective

To investigate the effects of human ASCs on the healing of cutaneous wounds in nude mice.

Methods

15-mm round full-thickness skin defects were generated on the back of BALB/c nude mice. The mice were divided into three groups for wound coverage: (i) human ASCs-populated collagen gel, (ii) human dermal fibroblasts-populated collagen gel, and (iii) collagen gel alone. Wound contraction was prevented with a splint method. Wound size was measured 10 days after injury. At 28 days histological analysis was performed.

Results

Both ASCs and dermal fibroblasts accelerated wound closure, but dermal fibroblasts were more effective than ASCs. At 28 days, the dermal portion of ASCs or dermal fibroblasts wound scars were thicker than collagen gel wound scars.

Conclusion

ASCs and dermal fibroblasts stimulate cutaneous wound healing and improve scar thickness.     

Sox9 Increases the Proliferation and Colony-forming Activity of Outer Root Sheath Cells Cultured In Vitro

Background

β-catenin plays a pivotal role in hair follicle development and hair growth cycle.

Objective

The aim of this study was to identify β-catenin-regulated genes in cultured human hair outer root sheath (ORS) cells.

Methods

Primary cultured ORS cells were transduced with recombinant adenovirus expressing N-terminal truncated β-catenin (constitutive active form), and β-catenin-regulated genes were identified.

Results

Overexpression of the constitutively active form of β-catenin led to induction of Sox9 expression at both mRNA and protein levels. To investigate the potential role of Sox9, we made the recombinant adenovirus expressing green fluorescent protein-tagged Sox9, and then transduced into cultured ORS cells. Interestingly, Sox9 induced the expression of keratin 15, increased the proliferation of ORS cells in vitro, and enhanced colony-forming activity.

Conclusion

Our results suggest that Sox9 is a β-catenin-regulated gene in ORS cells, and has potential importance in the regulation of hair follicle homeostasis.     

The Immunohistochemical Patterns of the ��-Catenin Expression in Pilomatricoma

Background

Pilomatricoma is a benign follicular tumor that is composed of basaloid cells, transitional cells and shadow cells. β-Catenin is a 92-kDa protein, and it plays important roles in cell-cell adhesion at the cell membrane and signal transduction in the nucleus. β-Catenin has recently been shown to play an important role in the formation of hair follicle-related tumors, including pilomatricoma. However, the pattern and the intracellular localization of the β-Catenin expression are still controversial.

Objective

We wanted to evaluate the pattern and the intracellular localization of the β-Catenin expression in pilomatricoma by performing immunohistochemical staining.

Methods

Twenty-seven paraffin-embedded tissue samples that were diagnosed as pilomatricoma were immunohistochemically stained with β-Catenin antibody.

Results

Basaloid cells were found 15 samples of the total 27 pilomatricomas. All (15/15) of the basaloid cells strongly expressed β-Catenin, but the transitional cells and the shadow cells did not. In the basaloid cells, the nuclei and membranes showed prominent β-Catenin immunoreactivities, but the cytoplasm showed weak β-Catenin immunoreactivity.

Conclusion

This study confirmed that the nucleus and membrane of all the basaloid cells in the pilomatricomas showed a strong β-Catenin expression, but the transitional cells and shadow cells showed negative β-Catenin immunoreactivity.     

Differential Expression of TGF-β Isoforms in Human Kerationocytes by Narrow Band UVB

Background

Transforming growth factor-β (TGF-β), a multifunctional growth factor, has three isoforms: TGF-β1, TGF-β2, and TGF-β3. Different isoforms of TGF-β are associated with different proliferation and differentiation states of the epidermis. Narrow band ultraviolet B (NBUVB) emits a concentrated UVB source of 311 nm. NBUVB 1,000 mJ/cm² induces apoptosis in approximately 50% of keratinocytes.

Objective

The purpose of this study was to evaluate whether irradiation with NBUVB would alter the expression and production of TGF-β1, 2, and 3.

Methods

We measured TGF-β1, 2, and 3 mRNA and TGF-β1 and 2 protein levels at 800, 1,000, and 1,200 mJ/cm² for 24 hours and 48 hours.

Results

TGF-β1 mRNA levels were increased at both 24 hr and 48 hr, TGF-β2 mRNA levels were decreased at both 24 hr and 48 hr, and TGF-β3 mRNA levels were increased at 24 hr and similar to control at 48 hr. TGF-β1 protein levels were increased at 48 hr but decreased at 24 hr. TGF-β2 protein levels were decreased at both 24 hr and 48 hr.

Conclusion

The results suggest a possible role for TGF-β1 after NBUVB irradiation and opposing roles for TGF-β1 and TGF-β2 isoforms in NBUVB irradiation.     

HR-1 Mice: A New Inflammatory Acne Mouse Model

Background

There is no appropriate in vivo animal model that reflects the inflammatory response of human acne.

Objective

This study investigated the effect of Propionibacterium acnes on the development of inflammatory acne-like lesions in four mouse strains with different degrees of immune response for the development of an optimal mouse model of inflammatory acne.

Methods

Human P. acnes suspensions (10⁸ and 10⁹ colony forming unit [CFU]/µl) were injected into the backs of HR-1, BALB/c, vitamin D receptor-knockout (VDR k/o), and severe combined immunodeficiency disease mice. Inflammation levels were evaluated two weeks after injection of P. acnes suspensions. In addition, histopathological examination and immunohistochemical staining of the expressions of inflammatory biomarkers (i.e., CD4+/CD8+ T lymphocytes, neutrophils, myeloperoxidase, interleukin-1β, matrix metalloprotease (MMP)-2, MMP-3, MMP-9, toll-like receptor (TLR)-2, LL-37, and integrin α6) were performed on tissue specimens.

Results

The HR-1 mouse strain exhibited the most remarkable inflammatory reaction with epithelial proliferation and microcomedone-like cyst formation. HR-1 mice also demonstrated aberrant integrin expression in the epidermis around both inflamed lesions and newly formed microcomedones. These findings were more prominent in the group receiving 10⁹ CFU/µl P. acnes than 10⁸ CFU/µl. MMP-9 expression in HR-1 mice was also upregulated around the microcomedone-like cysts. Finally, expression levels of TLR-2 and LL-37 were higher in HR-1 and BALB/c mice than the VDR k/o and SCID mice strains.

Conclusion

P. acnes induces acneiform inflammation with small microcomedones in HR-1 mice. Therefore, the HR-1 mouse strain represents a good candidate for the development of a new inflammatory acne mouse model.     

A Novel Compound Rasatiol Isolated from Raphanus sativus Has a Potential to Enhance Extracellular Matrix Synthesis in Dermal Fibroblasts

Background

The fibrous proteins of extracellular matrix (ECM) produced by dermal fibroblast contributes to the maintenance of connective tissue integrity.

Objective

This study is carried out to identify the bioactive ingredient from natural products that enhances ECM production in dermal fibroblasts.

Methods

Bioassay-directed fractionation was used to isolate the active ingredient from natural extracts. The effects of rasatiol (isolated from Raphanus sativus) on ECM production in primary cultured human dermal fibroblasts was investigated by enzyme linked immunosorbent assay and western blot analysis.

Results

Rasatiol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen, fibronectin and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was remarkably increased by rasatiol, indicating that enhanced ECM production is linked to the activation of intracellular signaling cascades.

Conclusion

These results indicate that rasatiol stimulates the fibrous components of ECM production, and may be applied to the maintenance of skin texture.