ABSTRACT
Pulsed electrical stimulation (PES) is known to affect cellular activities. We previously found PES to human dermal fibroblasts (HFs) promoted platelet-derived growth factor subunit A (PDGFA) gene expression, which enhanced proliferation. In this study, we investigated PES effects on fibroblast collagen production and differentiation into myofibroblasts. HFs were electrically stimulated at 4800 Hz and 5 V for 60 min. Imatinib, a specific inhibitor of PDGF receptors, was treated before PES. After 6 h of PES, PDGFA, α-smooth muscle actin (α-SMA), and collagen type I α1 chain gene expressions were upregulated in PES group. Imatinib suppressed the promoted expression except for PDGFA. Immunofluorescence staining and enzyme-linked immunosorbent assay showed the production of α-SMA and collagen I was enhanced in PES group but suppressed in PES + imatinib group at 48 h after PES. Therefore, PES promotes the production of α-SMA and collagen I in fibroblasts, which is triggered by PDGFA that is upregulated early after PES.
Subject(s)
Actins , Collagen Type I , Electric Stimulation , Fibroblasts , Platelet-Derived Growth Factor , Humans , Collagen Type I/metabolism , Collagen Type I/genetics , Actins/metabolism , Actins/genetics , Fibroblasts/metabolism , Fibroblasts/drug effects , Platelet-Derived Growth Factor/metabolism , Imatinib Mesylate/pharmacology , Cell Differentiation/drug effects , Skin/metabolism , Skin/cytology , Cells, Cultured , Gene Expression Regulation/drug effects , Dermis/cytology , Dermis/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Receptors, Platelet-Derived Growth Factor/genetics , Up-RegulationABSTRACT
Several collagen types are important for maintaining skin structure and function. Previous reports show that l-hydroxyproline (Hyp), N-acetyl-l-hydroxyproline (AHyp), and l-alanyl-l-glutamine (Aln-Gln) are biological active substances with collagen synthesis-promoting effects. In this study, we combined the promotive effects of pulsed electrical stimulation (PES) with three amino acid derivatives in human dermal fibroblasts. Fibroblasts were exposed to PES with a 4,800 Hz pulse frequency and a voltage at 1 or 5 V for 15 min. The gene expression of type I and III collagen (fibrillar collagen), type IV and VII collagen (basement membrane collagen and anchoring fibril collagen) were measured by RT-PCR 48 h after PES. PES alone promoted the expression of COL1A1 and COL3A1 at 5 V but did not alter that of COL4A1 and COL7A1. Each AAD and the AAD mixture promoted the expression of COL4A1 and COL7A1 but either repressed, or did not alter, that of COL1A1 and COL3A1. Compared to treatment with each AAD, PES at 5 V with Hyp promoted the expression of COL1A1 and COL3A1, enhanced COL3A1 expression with AHyp, and stimulated COL3A1 expression with Aln-Gln, while COL4A1 and COL7A1 expressions were not affected. PES and the AAD mixture significantly promoted COL4A1 expression in a voltage-dependent manner, and COL1A1 and COL3A1 demonstrated a similar but nonsignificant trend, whereas COL7A1 expression was not affected. The combination of PES with each AAD or the AAD mixture may improve skin structure and function by increasing the expression of basement membrane collagen and dermal fibrillar collagen.
ABSTRACT
Human dermal fibroblast proliferation plays an important role in skin wound healing, and electrical stimulation (ES) promotes skin wound healing. Although the use of ES for skin wound healing has been investigated, the mechanism underlying the effects of ES on cells is still unclear. This study examined the effects of pulsed electrical stimulation (PES) on human dermal fibroblasts. Normal adult human dermal fibroblasts were exposed to a frequency of 4800 Hz, voltage of 1-5 V, and PES exposure time of 15, 30, and 60 min. Dermal fibroblast proliferation and growth factor gene expression were investigated for 6-48 h post PES. Dermal fibroblast proliferation significantly increased from 24 to 48 h post PES at a voltage of 5 V and PES exposure time of 60 min. Under the same conditions, post PES, platelet-derived growth factor subunit A (PDGFA), fibroblast growth factor 2 (FGF2), and transforming growth factor beta 1 (TGF-ß1) expression significantly increased from 6 to 24 h, 12 to 48 h, and 24 to 48 h, respectively. Imatinib, a specific inhibitor of platelet-derived growth factor receptor, significantly inhibited the proliferation of dermal fibroblasts promoted by PES, suggesting that PDGFA expression, an early response of PES, was involved in promoting the cell proliferation. Therefore, PES at 4800 Hz may initially promote PDGFA expression and subsequently stimulate the expression of two other growth factors, resulting in dermal fibroblast proliferation after 24 h or later. In conclusion, PES may activate the cell growth phase of wound healing.
Subject(s)
Dermis/metabolism , Electric Stimulation , Fibroblasts/metabolism , Gene Expression Regulation , Intercellular Signaling Peptides and Proteins/metabolism , Adult , Aged , Cell Proliferation , Fibroblast Growth Factor 2/metabolism , Gene Expression Profiling , Humans , Imatinib Mesylate/pharmacology , Platelet-Derived Growth Factor/metabolism , Time Factors , Transforming Growth Factor beta1/metabolism , Wound HealingABSTRACT
Exposure to UV radiation to human skin up-regulates the synthesis of matrix metalloproteinase (MMP) family. Gelatinases are member of MMPs which have been suggested to play an important role in photoaging such as wrinkle formation. To inhibit gelatinase activity is regarded to be very important to keep healthy skin and to protect wrinkle formation. On the other hand, anti-photoaging agents are expected to be derived from natural resources, especially plants. Plant extracts having gelatinase-inhibitory effect that might be used as safe anti-photoaging ingredient were widely screened. An extract of rhizomes of Curcuma longa L. showed inhibitory effect of gelatinase activity. Curcuminoids and slight amount of compound, 6,11-dihydroxy-3-(4-hydroxy-3-mthoxyphenethyl)-7-[(E)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-3-butenyl]-10-methoxy-2-oxabicyclo[6.3.1.]dodeca-1(11),8(12),9-trien-5-yl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate (curcuminoid D) were isolated as the gelatinase-inhibitory components from methanol extract of rhizomes. The structure of curcuminoid D was determined by means of spectral data including 1H- and 13C-NMR, and IR. Curcumin exerted the enhancing effect on deposition of basement membrane component at dermal-epidermal junction in skin equivalent model. Topical application of cream containing turmeric extract significantly improved facial skin elasticity and decreased the number of gelatinase-positive stratum corneum clusters in human facial skins. These results indicated that turmeric is an effective ingredient to improve skin condition and to prevent skin from photoaging by suppressing activation of gelatinase chronically caused by UV.
Subject(s)
Curcuma/chemistry , Gelatinases/antagonists & inhibitors , Plant Extracts/pharmacology , Rhizome/chemistry , Skin/drug effects , Cells, Cultured , Curcumin/pharmacology , Humans , Infant, Newborn , Keratinocytes/drug effects , Keratinocytes/physiology , Male , Methanol/pharmacology , Protective Agents/pharmacology , Skin/radiation effects , Skin Physiological Phenomena/drug effects , Ultraviolet Rays/adverse effectsABSTRACT
Type IV collagen isolated from lens capsule without enzymatic treatment is known to form a gel under physiological condition and influences cellular activities. In case of human keratinocytes, the suppression of proliferation on reconstituted type IV collagen gels was reported in monolayer culture. In this study, we examined effects of type IV collagen isolated from porcine lens capsule on epidermal formation in human skin equivalents (HSEs). Type IV collagen aggregates were prepared under the culture condition and the aggregates suppressed keratinocyte proliferation in monolayer culture as well as the culture on the gels. In HSEs, type IV collagen aggregates were reconstituted on the surface of contracted collagen gels containing human dermal fibroblasts and the keratinocytes were then cultured on the aggregates for 14 days. Interestingly, in HSEs with type IV collagen aggregates, the BrdU-positive keratinocytes were increased and the thickness of the epidermal layer was around twice than that of control culture. Epidermal differentiation markers were expressed in the upper layer of the epidermis and the defined deposition of human basement membrane components were increased at the dermal-epidermal junction. These results indicate that the type IV collagen aggregates stimulate the proliferation of basal keratinocytes and improve the stratification of epidermal layers in HSEs.
Subject(s)
Collagen Type IV/physiology , Culture Techniques , Epidermis , Keratinocytes/physiology , Basement Membrane , Cell Proliferation , Cells, Cultured , HumansABSTRACT
To investigate sexual dimorphism and postnatal changes in skin collagen expression, mRNA levels of collagens and their regulatory factors in male and female skin were examined during the first 120 days of age by quantitative realtime PCR. Levels of mRNAs encoding extracellular matrices did not show any differences between male and female mice until day 15. Col1a1 and Col1a2 mRNAs noticeably increased at day 30 and remained at high levels until day 120 in male mice, while those in female mice remained at low levels during the period. Consistent with the mRNA expression, pepsin-soluble type I collagen contents in skin was very high in mature male as compared to female. Col3a1 mRNA in male mice also showed significantly high level at day 120 as compared to female. On the other hand, expression of mRNAs encoding TGF-ßs and their receptors did not show apparent sexual dimorphism although small significant differences were observed at some points. Castration at 60 days of age resulted in a significant decrease in type I collagen mRNA expression within 3 days, and noticeably decreased expression of all fibril collagen mRNAs examined within 14 days, while administration of testosterone tube maintained the mRNA expression at high levels. Despite the in vivo effect of testosterone, administration of physiological concentrations of testosterone did not affect fibril collagen mRNA expression in either human or mouse skin fibroblasts in vitro, suggesting that testosterone does not directly affect collagen expression in fibroblasts. In summary, present study demonstrated dynamic postnatal changes in expression of collagens and their regulatory factors, and suggest that testosterone and its effects on collagen expression are responsible for the skin sexual dimorphism but the effects of testosterone is not due to direct action on dermal fibroblasts.
Subject(s)
Collagen/genetics , Gene Expression Regulation, Developmental , Sex Characteristics , Skin/metabolism , Animals , Animals, Newborn , Castration , Collagen/metabolism , Cross-Linking Reagents/metabolism , Dermis/drug effects , Dermis/growth & development , Dermis/metabolism , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Female , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation, Developmental/drug effects , Humans , Infant, Newborn , Male , Mice, Inbred C57BL , RNA, Messenger/genetics , RNA, Messenger/metabolism , Skin/drug effects , Testosterone/pharmacologyABSTRACT
This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin-1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin-1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.
Subject(s)
Burns/therapy , Cicatrix/pathology , Elastin/physiology , Epithelium/growth & development , Fibrillin-1/physiology , Transplantation, Autologous , Wound Healing/physiology , Adolescent , Adult , Aged , Cells, Cultured , Elasticity/physiology , Female , Humans , Male , Middle Aged , Skin/pathology , Treatment Outcome , Young AdultABSTRACT
This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six-grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.
Subject(s)
Burns , Antigens, Differentiation , Autografts , Cell Differentiation , Cells, Cultured , Cicatrix , Epidermis , Filaggrin Proteins , Humans , Skin TransplantationABSTRACT
The renin-angiotensin system is known to be involved in skin remodeling and inflammation. Previously, we reported that ultraviolet B (UVB) irradiation enhanced angiotensin-converting enzyme (ACE) expression and angiotensin II levels in hairless mouse skin, and an ACE inhibitor, enalapril maleate (EM), accelerated repair of UVB-induced wrinkles. In this study, we analyzed gene expression profiles by DNA microarray and protein distribution patterns using an immunofluorescence method to clarify the process of EM-accelerated wrinkle repair in UVB-irradiated hairless mouse skin. In the microarray analysis, we detected EM-induced up-regulation of various extracellular matrix (ECM)-related genes in the UVB-irradiated skin. In the immunofluorescence, we confirmed that type I collagen α1 chain, fibrillin 1, elastin and dystroglycan 1 in the skin decreased after repeated UVB irradiation but staining for these proteins was improved by EM treatment. In addition, ADAMTS2 and MMP-14 also increased in the EM-treated skin. Although the relationship between these molecules and wrinkle formation is not clear yet, our present data suggest that the molecules are involved in the repair of UVB-induced wrinkles.
ABSTRACT
Epidermal-dermal interaction plays important roles in physiological events such as wound healing. In this study, we examined a double paracrine mechanism between keratinocytes and fibroblasts through interleukin-1 (IL-1) and an IL-1-induced inflammatory mediator prostaglandin E2 (PGE2) using the skin equivalent. The epidermal layer of the skin equivalent expressed high levels of IL-1α mRNA (IL1A mRNA) and relatively low levels of IL-1ß mRNA (IL1B mRNA). IL1A mRNA was not detected in fibroblasts. Fibroblasts also expressed low but not negligible levels of IL1B mRNA only in the presence of keratinocytes. Expression of prostaglandin-endoperoxide synthase 2 mRNA (PTGS2â mRNA) and production of PGE2 in three-dimensionally cultured fibroblasts were noticeably stimulated by co-culture with keratinocytes, whereas PTGS2â mRNA expression in the epidermal layer was very low. In addition, hydroxyprostaglandin dehydrogenase 15-(NAD) mRNA was highly expressed in keratinocytes but not in fibroblasts, and exogenous IL-1ß stimulated PTGS2â mRNA expression in the dermal equivalent. The thickness of the epidermal layer and the number of MKI67-positive keratinocytes in the skin equivalent were decreased by treatment with indomethacin, and the decrease recovered when exogenous PGE2 was added. These results indicate that keratinocytes stimulate their own proliferation through a double paracrine mechanism mediated by IL-1 and PGE2.
Subject(s)
Dinoprostone/metabolism , Epidermis/metabolism , Fibroblasts/metabolism , Keratinocytes/metabolism , Wound Healing , Cell Proliferation , Cells, Cultured , Coculture Techniques , Dinoprostone/pharmacology , Humans , RNA, Messenger/metabolismABSTRACT
BACKGROUND/PURPOSE: Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent. METHODS: We developed a novel device to apply repeated folding stress to the dermal equivalent. After applying the mechanical stress, morphological changes of the dermal equivalent and expression of several genes related to extracellular matrix turn over and cell contraction were examined. RESULTS: The repeated folding stress induced a noticeable decrease in the width of the dermal equivalent. The mechanical stress altered orientations of collagen fibrils. Hydroxyproline contents, dry weights and cell viability of the dermal equivalents were not affected by the mechanical stress. On the other hand, Rho-associated coiled-coil-containing kinase (ROCK) specific inhibitor Y27632 completely suppressed the decrease in the width of the dermal equivalent. CONCLUSION: The present results revealed that either degradation of collagen or changes in the number of cells were not responsible for the decrease in the width of the dermal equivalent and indicate that the repeated mechanical stress induces unidirectional contraction in the dermal equivalent through the RhoA-ROCK signaling pathway.
Subject(s)
Collagen Type I/metabolism , Fibroblasts/cytology , Fibroblasts/physiology , Mechanotransduction, Cellular/physiology , Skin, Artificial , Cells, Cultured , Compressive Strength/physiology , Elastic Modulus/physiology , Humans , Stress, Mechanical , Tensile Strength/physiology , ViscosityABSTRACT
Angiotensin-converting enzyme (ACE) activity and angiotensin II signaling regulate cell proliferation, differentiation, and tissue remodeling, as well as blood pressure, while in skin, angiotensin II signaling is involved in wound healing, inflammation, and pathological scar formation. Therefore, we hypothesized that angiotensin II is also involved in photoaging of skin. In this study, we examined the effect of enalapril maleate, an ACE inhibitor, on recovery of wrinkled skin of hairless mice exposed to long-term UVB irradiation. Immunohistochemical observation revealed that expression of ACE, angiotensin II, and angiotensin II type 1 (AT1) and type 2 (AT2) receptors in the skin was increased after UVB irradiation (3 times/week at increasing intensities for 8 weeks). Administration of enalapril maleate (5 times/week for 6 weeks, starting 1 week after 10-week irradiation) accelerated recovery from UVB-induced wrinkles, epidermal hyperplasia and epidermal barrier dysfunction, as compared with the vehicle control. Our results indicate that ACE and angiotensin II activity are involved in skin photoaging, and suggest that ACE inhibitor such as enalapril maleate may have potential for improvement of photoaged skin.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Enalapril/pharmacology , Epidermis/radiation effects , Skin Aging/drug effects , Angiotensin II/metabolism , Animals , Epidermis/drug effects , Epidermis/metabolism , Male , Mice , Mice, Hairless , Recovery of Function/drug effects , Signal Transduction/drug effects , Signal Transduction/radiation effects , Ultraviolet Rays/adverse effectsABSTRACT
Although normal human keratinocytes are known to migrate toward the cathode in a direct current (DC) electric field, other effects of the electric stimulation on keratinocyte activities are still unclear. We have investigated the keratinocyte differentiation under monodirectional pulsed electric stimulation which reduces the electrothermal and electrochemical hazards of a DC application. When cultured keratinocytes were exposed to the electric field of 3 V (ca. 100 mV/mm) or 5 V (ca. 166 mV/mm) at a frequency of 4,800 Hz for 5 min a day for 5 days, cell growth under the 5-V stimulation was significantly suppressed as compared with the control culture. Expression of mRNAs encoding keratinocyte differentiation markers such as keratin 10, involucrin, transglutaminase 1, and filaggrin was significantly increased in response to the 5-V stimulation, while the 3-V stimulation induced no significant change. After the 5-V stimulation, enhanced immunofluorescent stainings of involucrin and filaggrin were observed. These results indicate that monodirectional pulsed electric stimulation induces the keratinocyte differentiation with growth arrest.
Subject(s)
Cell Differentiation , Keratinocytes/physiology , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Proliferation , Cells, Cultured , Electric Stimulation , Filaggrin Proteins , Gene Expression , Humans , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
BACKGROUND: Keratinocytes in the hair follicle bulge region have a high proliferative capacity, with characteristics of epithelial stem cells. This cell population might thus be an ideal source for generating the interfollicular epidermis in a canine skin equivalent. HYPOTHESIS/OBJECTIVES: This study was designed to determine the ability of canine hair follicle bulge cell-enriched keratinocytes to construct canine living skin equivalents with interfollicular epidermis in vitro. ANIMALS: Four healthy beagle dogs from a research colony. METHODS: Bulge cell-enriched keratinocytes showing keratin 15 immunoreactivity were isolated from canine hair follicles and cultured on dermal equivalent containing canine fibroblasts. Skin equivalents were subjected to histological, immunohistochemical, western blot and RT-PCR analyses after 10-14 days of culture at the air-liquid interface. RESULTS: The keratinocyte sheets showed an interfollicular epidermal structure comprising four to five living cell layers covered with a horny layer. Immunoreactivities for keratin 14 and desmoglein 3 were detected in the basal and immediate suprabasilar layers of the epidermis, while keratin 10 and desmoglein 1 occurred in more superficial layers. Claudin 1 immunoreactivity was seen in the suprabasalar layer of the constructed epidermis, and filaggrin monomers and loricrin were detected in the uppermost layer. Basal keratinocytes in the skin equivalent demonstrated immunoreactivity to antibodies against basement membrane zone molecules. CONCLUSIONS AND CLINICAL IMPORTANCE: A bulge stem cell-enriched population from canine hair follicles formed interfollicular epidermis within 2 weeks in vitro, and thus represents a promising model for regenerative therapy of canine skin.
Subject(s)
Cell Culture Techniques/veterinary , Dog Diseases/therapy , Hair Follicle/cytology , Keratinocytes/physiology , Skin Diseases/veterinary , Tissue Engineering/veterinary , Animals , Dogs , Skin Diseases/therapy , Tissue Engineering/methodsSubject(s)
Ascorbic Acid Deficiency/genetics , Ascorbic Acid Deficiency/metabolism , Atrophy/etiology , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Epidermis/pathology , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Skin Diseases/pathology , Skin Pigmentation/radiation effects , Ultraviolet Rays , Animals , Atrophy/metabolism , Body Weight , Disease Models, Animal , Epidermis/metabolism , Mice , Mice, Knockout , Skin/pathologyABSTRACT
During mouse skin wound healing, mRNAs encoding IL-1, activins, and TGF-ßs significantly increased. To elucidate involvement of IL-1 in the regulation of activins and related factors in the wounded skin, human foreskin fibroblasts were stimulated with IL-1ß, and levels of mRNAs encoding activins, TGF-ßs, and follistatin family proteins were examined by quantitative real-time PCR. IL-1ß increased activin ßA (INHBA) and follistatin (FST) mRNA expression within 6 h. A p38 MAPK inhibitor, SB202190, a MAPK/ERK kinase inhibitor, U0126, and an nuclear factor κB pathway inhibitor, SC-514, significantly suppressed the IL-1ß-stimulated INHBA and FST mRNA expression. A prostaglandin-endoperoxide synthase inhibitor indomethacin, a potent inhibitor of prostaglandin E(2) (PGE(2)) synthesis, also significantly suppressed the IL-1ß-stimulated INHBA but not FST mRNA expression. Furthermore, stimulation of fibroblasts with PGE(2) significantly increased INHBA mRNA. The PGE(2)-induced INHBA mRNA expression was significantly suppressed by U0126 and a protein kinase C inhibitor, Gö 6983. Although IL-1ß stimulated FST mRNA in an acute phase, long-term exposure of fibroblasts to IL-1ß revealed time-dependent stimulatory and inhibitory effects of IL-1ß on FST mRNA expression. On the other hand, coculture with keratinocytes significantly increased INHBA mRNA expression in dermal equivalents. In summary, the present study indicates that the p38 MAPK, the MAPK/ERK kinase, the nuclear factor κB pathway, and PGE(2) mediate the effects of IL-1ß on INHBA mRNA expression. Furthermore, it is indicated that keratinocyte-derived factor of factors stimulate INHBA mRNA expression during wound healing.
Subject(s)
Dinoprostone/physiology , Inhibin-beta Subunits/genetics , Interleukin-1beta/pharmacology , MAP Kinase Signaling System/physiology , NF-kappa B/physiology , RNA, Messenger/analysis , Animals , Cells, Cultured , Cyclooxygenase 2/genetics , Fibroblasts/metabolism , Genes, fos , Mice , Mice, Inbred C57BL , Protein Kinase C/physiology , Wound HealingABSTRACT
Coenzyme Q10 (CoQ10), which has both energizing and anti-oxidative effects, is also reported to have antiaging action, e.g., reducing the area of facial wrinkles. However, the mechanism of its anti-aging activity is not fully established. Here, we examined the effect of CoQ10 on human dermal and epidermal cells. CoQ10 promoted proliferation of fibroblasts but not keratinocytes. It also accelerated production of basement membrane components, i.e., laminin 332 and type IV and VII collagens, in keratinocytes and fibroblasts, respectively; however, it had no effect on type I collagen production in fibroblasts. CoQ10 also showed protective effects against cell death induced by several reactive oxygen species in keratinocytes, but only when its cellular absorption was enhanced by pretreatment of the cells with highly CoQ10-loaded serum. These results suggest that protection of epidermis against oxidative stress and enhancement of production of epidermal basement membrane components may be involved in the antiaging properties of CoQ10 in skin.
Subject(s)
Basement Membrane/physiology , Cell Death/drug effects , Dermis/metabolism , Epidermis/metabolism , Oxidative Stress/drug effects , Ubiquinone/analogs & derivatives , Amidines/pharmacology , Cell Adhesion Molecules/biosynthesis , Cell Proliferation/drug effects , Collagen Type IV/biosynthesis , Collagen Type VII/biosynthesis , Dermis/cytology , Epidermal Cells , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Hydrogen Peroxide/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Male , Ubiquinone/physiology , KalininABSTRACT
Senescence marker protein-30 (SMP30) is a gluconolactonase required for vitamin C (VC) synthesis. We examined effects of VC deficiency on the mouse skin using SMP30 knockout (KO) mice. SMP30 KO or wild type male mice were weaned around day 30 of age, and fed VC-deficient diet. They were given either VC water or control water. VC deficiency for 36 days did not affect skin hydroxyproline contents, while VC deficiency for 60 days decreased the hydroxyproline levels. Levels of some collagen mRNAs were different among the groups, but did not correlate with skin VC levels. The epidermis was morphologically abnormal in VC-deficient SMP30 KO mouse at 60 days after the weaning. Interestingly, the hair cycle was not synchronized among the groups. These data suggest low susceptibility of the mouse skin to VC deficiency and involvement of VC in the regulation of keratinocyte function and hair cycle in vivo.
Subject(s)
Ascorbic Acid Deficiency/pathology , Ascorbic Acid/metabolism , Keratinocytes/metabolism , Skin/pathology , Animals , Calcium-Binding Proteins/genetics , Collagen/biosynthesis , Collagen/genetics , Hair/physiology , Intracellular Signaling Peptides and Proteins/genetics , Keratinocytes/pathology , Male , Mice , Mice, Knockout , Skin/metabolismABSTRACT
In adult mouse, the mRNA corresponding to the alpha1 chain of type XII collagen (alpha 1(XII)) is predominantly detected in the bone. Additionally, murine osteoblastic cells, MC3T3-E1, increased the mRNA level of alpha 1(XII) response to the mechanical strain in the stretch culture system. Cyclic stretch stress resulted in a threefold increase in mRNA level of alpha 1(XII) as compared to the control experiment in MC3T3-E1. Transient transfection assays employing a reporter construct, together with site-directed mutagenesis studies, suggested that the AP-1 binding site in the first exon of mouse alpha 1(XII) gene is important for stretch stress-mediated upregulation of alpha 1(XII) expression. Electrophoretic mobility shift assay and associated antibody supershift experiments showed that stretch stress promotes the binding of c-Jun and JunD. Further chromatin immunoprecipitation experiments confirmed the participation of these transcription factors in the region. Also, the exogenous induction of the dominant negative form of c-Jun canceled the effect of stretch stress on the stimulation of the alpha 1(XII) gene. Here, we reported a potential responsive element to the stretch stress in mouse alpha 1(XII) gene. These data will provide new information on the mechanical strain-mediated transcriptional control of alpha 1(XII)-mediated fibrillogenesis in the bone.
Subject(s)
Collagen Type XII/genetics , Osteoblasts/metabolism , Stress, Mechanical , Up-Regulation , Animals , Base Sequence , Bone and Bones/cytology , Bone and Bones/metabolism , Cell Line , Cells, Cultured , Exons , Mice , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Expression of procollagens (Col1a1/2, Col3a1, Col4a1/2, Col5a1/2) and fibronectin 1 (Fn1) in the mouse fetal placental tissue was examined during the second half of pregnancy. Ribonuclease protection assays (RPAs) revealed that levels of these mRNAs noticeably increased between Days 10 and 14 of pregnancy, and they remained at relatively constant levels thereafter. In situ hyridization showed that Col1a1 and Col4a1 mainly localized in the labyrinth, whereas Fn1 was expressed mainly in the spongiotrophoblast. Since members of the transforming growth factor-beta (TGFB) superfamily are involved in the regulation of extracellular matrix (ECM) expression in various tissues, mRNA levels of TGFB family members and their binding proteins were also examined by RPAs. Transforming growth factor-beta1-3 (Tgfb1-3), activin subunits (Inhba, Inhbb), follistatin (Fst), and follistatin-like 3 (Fstl3) were expressed in the placenta, whereas significant expression of myostatin (Mstn) was not detected. Although the expression patterns of Tgfb1-3 and Inhba in the placenta suggest possible involvement of TGFBs and activin A in the regulation of placental ECM expression, neither TGFBs nor activin A affected ECM mRNA levels in vitro. On the other hand, hypoxia significantly decreased Col1a1/2 and Col4a1/2 mRNAs in cultured placental cells, and a high-glucose condition significantly increased Col1a1 and Col3a1 mRNAs. Fn1 expression was increased under the high-glucose condition, although hypoxia also increased Fn1 expression to a lesser degree. These data suggest that an increase in oxygen tension and nutrient supply during placentation rather than TGFB family members may be responsible for the increase in the placental ECM mRNA expression.