Han-Sun Chiang

Protective effects of (−)-epicatechin-3-gallate on UVA-induced damage in HaCaT keratinocytes

Abstract(−)-Epigallocatechin-3-gallate (EGCG), a constituent of green tea, has been extensively studied and shown to be a powerful antioxidant protecting skin cells against photodamage. In this study, however, we demonstrated that another gallated catechin, (−)-epicatechin-3-gallate (ECG), was also able to protect human keratinocytes against damage induced by ultraviolet A (UVA) light. We found that ECG dose-dependently inhibited UVA-induced keratinocyte death as determined by cell viability assay. Moreover, ECG had similar potency to EGCG in inhibiting UVA-induced cell death. Therefore, the mechanism of action of ECG was further investigated. As assayed by flow cytometry, UVA-induced hydrogen peroxide (H2O2) production in keratinocytes was inhibited by ECG in a concentration-dependent manner, suggesting that ECG can act as a free radical scavenger while keratinocytes were photodamaged. The scavenging effect of ECG was confirmed by the fact that ECG treatment attenuated cell damage induced by H2O2 and hypoxanthine-xanthine oxidase. In a parallel experiment, UVA-induced activation of extracellular signal-regulated kinase in keratinocytes was blocked by ECG. We provided here the first evidence that ECG is a potent protectant that protects keratinocytes from photodamage. Because ECG is abundant in green tea, we believe that this compound is beneficial for skin care.

(-)-Epicatechin-3-gallate, a green tea polyphenol is a potent agent against UVB-induced damage in HaCaT keratinocytes.

(-)-Epicatechin-3-gallate (ECG) is a polyphenolic compound similar to (-)-epigallocatechin-3-gallate (EGCG) which is abundant in green tea. Numerous workers have proposed that EGCG protects epidermal cells against UVB-induced damage. However, little has been known about whether ECG protects keratinocytes against UVB-induced damage. We decided to investigate the protective effects and underlying mechanisms of ECG on UVB-induced damage. Cell viability was determined by the MTT assay. Activation of ERK1/2, p38 and JNK was analyzed by Western blotting. Intracellular H2O2 production and DNA content was analyzed by flow cytometry. Lipid peroxidation was assayed by colorimetry. In our study, we found that ECG dose-dependently attenuated UVB-induced keratinocyte death. Moreover, ECG markedly inhibited UVB-induced cell membrane lipid peroxidation and H2O2 generation in keratinocytes, suggesting that ECG can act as a free radical scavenger when keratinocytes were photodamaged. In parallel, H2O2-induced the activation of ERK1/2, p38 and JNK in keratinocytes could be inhibited by ECG. UVB-induced pre-G1 arrest leading to apoptotic changes of keratinocytes were blocked by ECG. Taken together, we provide here evidence that ECG protects keratinocytes from UVB-induced photodamage and H2O2-induced oxidative stress, possibly through inhibition of the activation of ERK1/2, p38 and JNK and/or scavenging of free radicals.

(−)‐Epigallocatechin‐3‐gallate, a polyphenolic compound from green tea, inhibits fibroblast adhesion and migration through multiple mechanisms

It is increasingly evident that the stromal cells are involved in key metastatic processes of melanoma and some malignant solid tumors. (−)‐Epigallocatechin‐3‐gallate (EGCG), a polyphenolic compound from green tea, has been shown to have anti‐tumor activity, inhibiting adhesion, migration, and proliferation of tumor cells. However, little attention has been paid on its effects on stromal cells. In the present study, we determined the effects of EGCG on stromal fibroblasts. We showed that fibroblast adhesion to collagen, fibronectin, and fibrinogen were inhibited by EGCG. One of the possible mechanisms is binding of EGCG to fibronectin and fibrinogen but not to collagen. We then focused how EGCG affected fibroblast adhesion to collagen. EGCG treatment attenuated the antibody binding to fibroblasts integrin α2β1, indicating EGCG may affect the expression and affinity of integrin α2β1. Moreover, intracellular H2O2 level was decreased by EGCG treatment, suggesting that the tonic maintenance of intracellular H2O2 may be required for cell adhesion to collagen. In parallel, collagen‐induced FAK phosphorylation, actin cytoskeleton reorganization in fibroblasts, migration and matrix metalloproteinase(s) (MMPs) activity were also affected by EGCG. Tubular networks formed by melanoma cells grown on three‐dimensional Matrigel were also disrupted when fibroblasts were treated with EGCG in a non‐contact coculture system. Taken together, we provided here the first evidence that EGCG is an effective inhibitor on behaviors of the stromal fibroblasts, affecting their adhesion and migration. The inhibitory activity of EGCG may contribute to its anti‐tumor activity. The findings and concepts disclosed here may provide important basis for a further experiment towards understanding tumor–stroma interaction.

E-cadherin and its downstream catenins are proteolytically cleaved in human HaCaT keratinocytes exposed to UVB

Abstract:  It has been reported that ultraviolet B (UVB) irradiation causes the loss of E‐cadherin of melanocytes, leading them to escape from neighboring keratinocytes during melanoma development. However, little has been paid on its effect on E‐cadherin of keratinocytes. In the present study we therefore focus on whether UVB affects expression of E‐cadherin‐catenin complex in human HaCaT keratinocytes. We found that E‐cadherin, β‐, and γ‐catenin but not α‐catenin were proteolytically cleaved in UVB‐irradiated HaCaT keratinocytes. The effect was only observed in keratinocyte undergoing apoptosis. Cleavage of β‐ and γ‐catenin was fully abolished by caspase‐3 and caspase‐8 inhibitors, whereas cleavage of E‐cadherin was inhibited by neither caspase nor metalloproteinase inhibitors. Functional analysis showed that the cleavage resulted in the disruption of the physical association between E‐cadherin and catenins, indicating that E‐cadherin signaling was compromised in UVB‐irradiated HaCaT keratinocytes. Because E‐cadherin in keratinocytes plays important roles in mediating cell–cell adhesion in epidermis of skin, the loss of E‐cadherin and signaling components in keratinocytes may lead to the disruption of skin integrity after UVB exposure.

The role of the septin family in spermiogenesis

Septins (full name: Septin; symbol name: SEPT) belong to a family of polymerizing GTP binding proteins that are required for many cellular functions, including membrane compartmentalization, vesicle trafficking, mitosis and cytoskeletal remodeling. Two of the 14 family members in the mammalian species, Septin12 and 14 are expressed specifically in the testis. In the mouse, knockout of Septin4 and Septin12 leads to male sterility with distinctive sperm pathology (defective annulus or bent neck). In humans, sperm with abnormal expression pattern of SEPT4, 7 and 12 are more prevalent in infertile men. How septin filament is assembled /dissembled and how the SEPT-related complex regulates spermatogenesis still await further investigation.

Inhibitory effect of lycopene on PDGF-BB-induced signalling and migration in human dermal fibroblasts: a possible target for cancer.

Tumours are complex tissues composed of both matrix proteins and stromal cells such as fibroblasts and inflammatory cells. Tumour progression is often the result of dynamic interactions between the tumour cells and their surroundings. Lycopene, a natural carotenoid that is abundant in tomato, has been shown to inhibit proliferation of several types of cancer cells through arrest of tumour cell-cycle progression, IGF-1 (insulin-like growth factor 1) signalling transduction, induction of apoptosis etc. However, in our recent study, we found that lycopene inhibited PDGF-BB (platelet-derived growth factor-BB)-induced signalling and cell migration in human cultured skin fibroblasts through a novel mechanism of action, i.e. direct binding to PDGF-BB. Trapping of PDGF by lycopene also compromised melanoma-induced fibroblast migration and attenuated signalling transduction in fibroblasts simulated by melanoma-derived conditioned medium, suggesting that lycopene may interfere with tumour-stroma interactions. The trapping activity of lycopene on PDGF suggests that it may act as an inhibitor on stromal cells, tumour cells and their interactions, which may contribute to its anti-tumour activity.

SEPT12-Microtubule Complexes Are Required for Sperm Head and Tail Formation

The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/− chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and β-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and β-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and β-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and β-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and β-tubulins, in SEPTIN12+/+/+/− chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.

Optimizing a Male Reproductive Aging Mouse Model by D-Galactose Injection

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging.

Loss of SLC9A3 decreases CFTR protein and causes obstructed azoospermia in mice

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major CFTR mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (SLC9A3) gene. SLC9A3 is a Na+/H+ exchanger, and depleted Slc9a3 in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of Slc9a3 knockout mice. Adult Slc9a3-/- mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, Slc9a3-/- mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in Slc9a3–/–epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The Slc9a3-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry SLC9A3 deletion but without major CFTR mutations.

RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs) through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16). RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration) by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1), were identified using co-immunoprecipitation (co-IP) and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS). We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.