Il-Hong Bae

The effect of epidermal growth factor (EGF) conjugated with low-molecular-weight protamine (LMWP) on wound healing of the skin

This study was designed to develop a skin permeable recombinant low-molecular-weight protamine (LMWP) conjugated epidermal growth factor (EGF) (rLMWP-EGF) by linking a highly positive charged LMWP to the N-terminal of EGF through genetic recombination. We evaluated its biological activity, skin permeability, and wound healing efficacy in vivo. The cDNA for rLMWP-EGF was prepared by serial polymerase chain reaction for encoding amino acids of LMWP to the vector for EGF. After expression and purification, recombinant EGF site-specifically conjugated with LMWP was obtained. The in vitro cell proliferation activity was well preserved after LMWP conjugation and was comparable to that of rEGF. rLMWP-EGF showed markedly improved permeability through the three-dimensional artificial human skin constructs, and the cumulative permeation of rLMWP-EGF across the excised mouse skin was about 11 times higher than that of rEGF. Topically applied rLMWP-EGF significantly accelerated the wound closure rate in full thickness as well as a diabetic wound model most probably due to its enhanced skin permeation. These findings demonstrate the therapeutic potential of rLMWP-EGF as a new topical wound healing drug and the site-specific conjugation of LMWP to peptides or proteins by genetic recombination as a useful method for preparing highly effective biomedicines.

Determination of spatial distribution of melamine-cyanuric acid crystals in rat kidney tissue by histology and imaging matrix-assisted laser desorption/ionization quadrupole time-of-flight mass spectrometry.

After the outbreak of acute renal failure associated with melamine-contaminated pet food, many attempts have been made to uncover the mechanism underlying the renal toxicity caused by melamine and melamine-related compounds. Using rat models, we investigated the renal crystal formation following the ingestion of a melamine-cyanuric acid mixture (M+CA, 1:1) to gain insight into the M+CA-induced renal toxicity. M+CA did not induce toxicity in precision-cut kidney slices, suggesting that M+CA does not have a direct nephrotoxicity. On the contrary, oral administration of M+CA for 3 days induced nephrotoxicity as determined by increased serum blood urea nitrogen and creatinine, reduced creatinine clearance, and enlarged kidneys in the animals treated with 50 mg/kg M+CA (melamine, 25 mg/kg, and cyanuric acid, 25 mg/kg; 2 of 10 animals) and 100 mg/kg M+CA (9 of 9 animals). While urine crystals were found in all animals treated with M+CA (25-100 mg/kg), histological examination revealed that renal crystals could be observed only in the kidneys of animals showing signs of nephrotoxicity. Remarkably, at 50 mg/kg M+CA, crystals were observed mainly in the medulla region of the kidney, while at 100 mg/kg, crystals were disseminated throughout the cortex and medulla regions. To further investigate the crystal formation by M+CA, matrix-assisted laser desorption/ionization quadrupole time-of-flight (MALDI-Q-TOF) imaging mass spectrometry detecting melamine distribution through monitoring the product ion (m/z 85, M + H) from melamine (m/z 127, M + H) was developed to directly obtain the image of melamine distribution in the kidney. The distribution image of melamine in kidney tissue confirmed that dense points of melamine were located only in the medulla region at 50 mg/kg M+CA, while at 100 mg/kg, they were disseminated widely from the cortex to medulla. These results demonstrated that M+CA ingestion could lead to crystal formation in kidney tubules along the osmotic gradient and that renal crystal formation is closely linked with M+CA-induced nephrotoxicity.

Liver X Receptor Activation Inhibits Melanogenesis through the Acceleration of ERK-Mediated MITF Degradation

Liver X receptors (LXRs) are nuclear receptors that act as ligand-activated transcription factors regulating lipid metabolism and inflammation. In the skin, activation of LXRs stimulates differentiation of keratinocytes and augments lipid synthesis in sebocytes. However, the function of LXRs in melanocytes remains largely unknown. We investigated whether LXR activation would affect melanogenesis. In human primary melanocytes, MNT-1, and B16 melanoma cells, TO901317, a synthetic LXR ligand, inhibited melanogenesis. Small interfering RNA (siRNA) experiments revealed the dominant role of LXRβ in TO901317-mediated antimelanogenesis. Enzymatic activities of tyrosinase were unaffected, but the expression of tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 was suppressed by TO901317. Expressions of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of melanogenesis, and cAMP-responsive element-binding activation were not affected. It is noteworthy that the degradation of MITF was accelerated by TO901317. Extracellular signal-regulated kinase (ERK) contributed to TO901317-induced antimelanogenesis, which was evidenced by recovery of melanogenesis with ERK inhibitor. Other LXR ligands, 22(R)-hydroxycholesterol (22(R)HC) and GW3965, also activated ERK and suppressed melanogenesis. The intermediary role of Ras was confirmed in TO901317-induced ERK phosphorylation. Finally, antimelanogenic effects of TO901317 were confirmed in vivo in UVB-tanning model in brown guinea pigs, providing a previously unreported line of evidence that LXRs may be important targets for antimelanogenesis.

Staphylococcus aureus inhibits terminal differentiation of normal human keratinocytes by stimulating interleukin-6 secretion

BACKGROUND Staphylococcus aureus (S. aureus) is found on the skin of approximately 90% of patients with atopic dermatitis and approximately 20% of apparently healthy subjects. S. aureus induces keratinocytes and immune cells to secrete immunoregulatory factors that cause epidermal barrier dysfunction in atopic skin. OBJECTIVE This study examined factors that cause epidermal permeability barrier dysfunction in skin colonized by S. aureus. METHODS We examined the effect of S. aureus on keratinocyte differentiation in the stratum corneum (SC) of in vivo skin, normal human keratinocytes (NHKs) and a reconstructed human epidermis (RHE) model. The fold change in expression of the terminal differentiation markers and the level of secreted cytokines were investigated. RESULTS The SC displayed decreased expression of keratin 10 (KRT 10). NHKs treated with S. aureus extracts increased expression of interleukin (IL)-6 and significantly reduced expression of the terminal differentiation markers KRT 1, KRT 10, loricrin (LOR), and filaggrin (FLG); however, the expression of basal layer markers (KRT 5, KRT 14) remained unchanged. Treatment of NHKs with an anti-IL-6 antibody in combination with IL-6 or the S. aureus extracts inhibited the decrease in KRT 10 mRNA or protein expression. After the RHEs were exposed to the S. aureus extracts, KRT 1 and KRT 10 protein levels decreased. CONCLUSIONS These findings suggest that S. aureus inhibits the terminal differentiation of keratinocytes by stimulating IL-6 secretion.

Evaluation of a Highly Skin Permeable Low-Molecular-Weight Protamine Conjugated Epidermal Growth Factor for Novel Burn Wound Healing Therapy

We evaluated the laser induced burn wound healing efficacy of a recombinant low-molecular-weight protamine conjugated epidermal growth factor (rLMWP-EGF). rLMWP-EGF was prepared by genetically combining LMWP with the N-terminal sequence of EGF; we obtained a homogeneous modified EGF without reduced biological activity. Because of the protein transduction domain of LMWP, rLMWP-EGF showed enhanced drug penetration across artificial skin constructs and excised mouse skin layers versus EGF and showed significantly improved burn wound healing efficacy, with accelerated wound closure and minimized eschar and scar formation, compared with EGF or no treatment. Histological examination also revealed that rLMWP-EGF permeated through the intact skin around the wound and facilitated residual epithelial cell proliferation in an integrated manner to reform an intact epidermis. Radiofrequency microwound formation was effective for reducing large hypertrophic scars formed after severe laser burning by collagen remodeling but rLMWP-EGF did not show a meaningful synergistic effect in burn scar reduction. However, rLMWP-EGF was helpful for forming skin with a more normal appearance and texture. Thus, rLMWP-EGF demonstrated therapeutic potential as a novel topical burn wound healing drug with no obvious toxic effect.

Bioprinting of biomimetic skin containing melanocytes

This study reports a three‐dimensional (3D) bioprinting technique that is capable of producing a full‐thickness skin model containing pigmentation. Multiple layers of fibroblast (FB)‐containing collagen hydrogel precursor were printed and crosslinked through neutralization using sodium bicarbonate, constituting the dermal layer. Melanocytes (MCs) and keratinocytes (KCs) were sequentially printed on top of the dermal layer to induce skin pigmentation upon subsequent air‐liquid interface culture. Histological analysis was performed not only to confirm the formation of distinct skin layers, but also to identify the presence of pigmentation. The bioprinted skin structure showed the dermal and epidermal layers as well as the terminal differentiation of the KC that formed the stratum corneum. Moreover, the MC‐containing epidermal layer showed freckle‐like pigmentations at the dermal‐epidermal junction, without the use of external ultraviolet light or chemical stimuli. The presented method offers the capability of producing engineered ephelides in biomimetic skin, thus rendering 3D bioprinting techniques as productive on‐demand options for the creation of skin models available for therapeutic or research use.

Determination of the key innate genes related to individual variation in carbon tetrachloride-induced hepatotoxicity using a pre-biopsy procedure

High inter-individual variation in chemical-induced liver injury is a frequent observation with many hepatotoxic chemicals, yet the mechanism underlying it remains poorly understood. Even with carbon tetrachloride (CCl(4)), a well-known model hepatotoxicant, substantial individual variations are observed in the severity of liver injury. Using microarray, many attempts have been made to identify the key genes in CCl(4)-induced liver injury but mostly, they examined the gene expression of liver after CCl(4) exposure, unable to dissect out the complicating factors from pathological changes secondary to liver injury. To more accurately identify the genes for the individual variation in CCl(4)-induced hepatotoxicity, we compared the innate gene expression of the individual liver samples pre-biopsied prior to CCl(4)-treatment with the severity of liver injury after CCl(4)-treatment. Effect of biopsy procedure and 3 week recovery period on liver function and gene expression were confirmed to be insignificant. Using this design, we found that the expression of genes associated with immunity and defense, lipid metabolism, transport and complement-mediated immunity, which are previously known to be suppressed by CCl(4)-treatment, were innately lower in the susceptible animals than resistant animals. Moreover, we demonstrated that the genes such as Gsta2, Sult2a1, Fgl1 and C6 were newly found to be innately lower in the susceptible animals to CCl(4)-hepatotoxicity. These naturally lower gene expression patterns were further confirmed by RT-PCR. We believe that this pre-biopsy design may provide a useful tool for understanding the cause of variability of hepatotoxicity and for the prediction and pre-screening of the susceptible individual to drug-induced hepatotoxicity.

Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes

Background: The molecular mechanisms underlying the non-genomic activities of testosterone in keratinocytes are unknown. Results: Testosterone stimulates Duox1 activity through GPRC6A leading to cell death in skin keratinocyte. Conclusion: These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis through Duox1-induced H2O2 generation. Significance: These results provide a novel signaling cascade of testosterone-mediated redox regulation in keratinocytes. Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca2+]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca2+]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1.

S100A7 (psoriasin) inhibits human epidermal differentiation by enhanced IL‐6 secretion through IκB/NF‐κB signalling

Psoriasin (S100A7), a member of the S100 protein family, is a well‐known antimicrobial peptide and a signalling molecule which regulates cellular function and is highly expressed in hyperproliferative skin conditions such as atopic dermatitis (AD) and psoriasis with disrupted skin barrier function. However, its role in epidermal differentiation remains unknown. We examined the effect of S100A7 on epidermal differentiation in normal human keratinocytes (NHKs) and on a reconstituted human epidermis model. When NHKs were exposed to disruptive stimuli such as Staphylococcus aureus, ultraviolet irradiation and retinoic acid, the secretion of S100A7 into the culture medium increased and the expression of epidermal differentiation markers decreased. Treatment of NHKs with S100A7 significantly inhibited epidermal differentiation by reducing the expression of keratin 1, keratin 10, involucrin and loricrin and by increasing the expression of abnormal differentiation markers (keratin 6 and keratin 16). We verified that the MyD88‐IκB/NF‐κB signal cascade was activated via RAGE after S100A7 treatment, resulting in the upregulation of interleukin‐6. Finally, we confirmed that S100A7 is a negative regulator of epidermal differentiation using a reconstituted human epidermis model. This study suggests that S100A7‐related signalling molecules could be potent targets for recovering skin barrier function in AD and psoriasis where S100A7 is accumulated excessively.

Compound K inhibits MMP-1 expression through suppression of c-Src-dependent ERK activation in TNF-α-stimulated dermal fibroblast.

Compound K (CK) is one of the major metabolites of ginsenosides exhibiting a variety of pharmacological properties such as anti‐ageing, anti‐oxidation and anti‐inflammatory activities. However, the protective efficacy of CK in abnormal skin conditions with inflammatory responses was not examined. Here, we investigated the effects of CK on matrix metalloproteinase‐1 (MMP‐1) and type I procollagen production in tumor necrosis factor‐α (TNF‐α)‐stimulated human skin fibroblasts HS68 cells and human skin equivalents. We found that CK suppressed MMP‐1 secretion and increased the level of reduced type I procollagen secretion, caused by the inhibition of extracellular signal‐regulated kinase (ERK) activation, but not p38 and c‐Jun N‐terminal kinase (JNK) activation in TNF‐α‐stimulated HS68 cells. Then, we focused on the involvement of the c‐Src and epidermal growth factor receptor (EGFR) as upstream signalling molecules for ERK activation by TNF‐α in HS68 cells. CK suppressed the phosphorylation of c‐Src/EGFR by TNF‐α, which led to the inactivation of downstream signalling molecules including AKT and MEK. In addition, CK suppressed AP‐1 (c‐jun and c‐fos) phosphorylation as downstream transcription factors of active ERK for MMP‐1 expression in TNFα‐stimulated HS68 cells. These results showed novel mechanisms by which CK inhibits TNF‐α‐induced MMP‐1 expression through the inactivation of c‐Src/EGFR‐dependent ERK/AP‐1 signalling pathway, resulting in the inhibition of collagen degradation in human fibroblast cells. Therefore, CK may be a promising protective agent for the treatment of inflammatory skin conditions such as skin ageing and atopic dermatitis.