Spectrum of mitochondrial DNA deletions within the common deletion region induced by low levels of UVB irradiation of human keratinocytes in vitro

We show that a single low-dose exposure of human epidermal keratinocytes (NHEK) to an FS20 light source in vitro can induce the formation of mitochondrial DNA deletions in a PCR detection assay. We used primer sets specifically designed to exclude amplification of segments containing the common deletion, but which could detect possibly lower abundance deletions generated within the same region of the mitochondrial genome. We characterized eight novel deletions of which six were generated from cut sites within, or adjacent to, short direct repeats. Two deletions involved cut sites in inverted tetrameric repeats; one of these also involved an insertion.     

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.     

UVB-induced apoptosis in normal human keratinocytes: role of the erbB receptor family

Exposure of human keratinocytes to ultraviolet B (UVB) light leads to the activation of a variety of cell-surface receptors; however, the biologic consequences of these activated receptors are still unclear. It was previously reported that inhibition of cellular tyrosine kinase activity suppressed UVB-dependent effects in human skin. We confirmed that the same suppression of UVB-induced apoptosis occurs in normal human keratinocytes grown in culture. Furthermore, we sought to determine the role of erbB receptor tyrosine kinases in human keratinocytes following UVB irradiation. Using a specific inhibitor of the erbB family of tyrosine kinase receptors, DAPH, we investigated the effects of UVB-dependent activation of these receptors on keratinocyte biology. The addition of DAPH to keratinocytes resulted in the concentration-dependent protection of UVB-induced apoptosis. The protection from apoptosis was not due to the induction of keratinocyte differentiation, the loss of keratinocyte viability, or inhibition of the proliferative potential of keratinocytes by DAPH. The effect of DAPH on apoptosis was specific for UVB as it had no effect on bleomycin-induced apoptosis. Furthermore, the inhibition of UVB-induced apoptosis could also be observed using neutralizing antibodies to either erbB1 or erbB2. Finally, we demonstrated that DAPH could also inhibit UVB-induced apoptosis in an epidermal organotypic model system. These studies suggest an important role for the erbB receptors in UVB-induced apoptosis of human keratinocytes.     

Corticotropin-releasing hormone affects cytokine production in human HaCaT keratinocytes

CRH cutaneous expression is significantly enhanced after exposure to various stimuli (Physiol Rev 2000, 80;979-1020). We evaluated the effect of CRH on cytokine production in HaCaT keratinocytes, a cell line shown to express CRH receptors coupled to cAMP activation and calcium-dependent transmission pathways. It is demonstrated for the first time that exogenously added CRH stimulates production of IL-6 and IL-11. It also inhibits production of IL-1beta and does not affect TNF-alpha production. Our results indicate that CRH function(s) during cutaneous stress may be mediated by differential effects on cytokine production.     

The decreased secretion of hyaluronan by older human fibroblasts under physiological conditions is mainly associated with the down-regulated expression of hyaluronan synthases but not with the expression levels of hyaluronidases

Although it has been reported that levels of hyaluronan are decreased in the dermis of aged skin, little is known about the cellular mechanism(s) underlying that hyaluronan deficiency. Since hyaluronan is produced by dermal fibroblasts and is secreted into the surrounding dermal tissues, we examined the secretion of hyaluronan by dermal fibroblasts and characterized its cellular mechanism using real-time RT-PCR and western blotting for its synthesizing and degrading enzymes, hyaluronan synthase and hyaluronidase, respectively. The secretion of hyaluronan by dermal fibroblasts derived from differently aged human donors, was higher in the younger human fibroblasts tested (0 and 19 years old) compared to the older human fibroblasts tested (39, 56 and 77 years old). The relative secretion levels of hyaluronan by the different human fibroblasts tested were attributable to the relative expression of hyaluronan synthases 1, 2, 3 but not hyaluronidases 1, 2 enzymes at the gene and protein levels among those fibroblasts. These findings indicate that the deficiency of hyaluronan in the aged dermis might result from the down-regulation in the potential of older human fibroblasts to secrete hyaluronan and that decrease in secretory potential is mainly associated with the down-regulated expression of hyaluronan synthases, especially hyaluronan synthase 2, but not with the expression levels of hyaluronidases.     

Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the α subunit of Gq protein (Gαq) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active Gαq (GαqQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of Gαq with shRNA in HaCaT human keratinocytes. Gαq was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase Cδ (PKCδ), and matrix metaloprotease-2 (MMP-2). Moreover, GαqQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that Gαq mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKCδ and MMP-2 in HaCaT cells.     

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.     

Expression of P450 enzymes in rat whole skin and cultured epidermal keratinocytes

The complement and level of expression of P450 enzymes in male Fischer F344 rat whole skin and cultured keratinocytes were investigated using a panel of mono-specific antibodies. In whole skin microsomal fraction, immunoreactive bands corresponding to CYP2B12, CYP2C13, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP3A2 were detected whereas CYP1A1, CYP1A2, and CYP2C12 were absent. Skin levels were all between 0.1% and 4.7% of those found in liver, except for CYP2D4, which was not detected in liver. Keratinocytes were isolated from rat skin, cultured for up to 42 days, and changes in the levels of CYP3A1, CYP3A2, and CYP2E1 determined. Levels were low in isolated keratinocytes, but they increased markedly in culture, reaching a maximum at 10-14 days, where they were similar to those found in fresh skin. This suggests that primary keratinocytes grown in culture for 10-14 days may provide a useful experimental model to study P450-catalysed metabolism of xenobiotics in skin.     

Up-regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17β-estradiol and testosterone for 8–48 h. Treatment with 17β-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17β-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.     

High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

ER signaling is activated to protect human HaCaT keratinocytes from ER stress induced by environmental doses of UVB

Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-Lα, an upstream signaling molecule of ER stress, decreased at 1-4 h after 10 mJ/cm² irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm² UVB at 4 h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) –mediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures.     

Functional vanilloid receptors in cultured normal human epidermal keratinocytes

Vanilloid receptor subtype 1, VR1, is an ion channel that serves as a polymodal detector of pain-producing chemicals such as capsaicin and protons in primary afferent neurons. Here we showed that both capsaicin and acidification produced elevations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured human epidermal keratinocytes. The capsaicin- and acidification-evoked increases in [Ca(2+)](i) were inhibited by capsazepine, an antagonist to VR1. VR1-like immunoreactivity was observed in the cells. These findings suggest that functional VR1-like protein is present and functions as a sensor against noxious chemical stimuli, such as capsaicin or acidification, in epidermal keratinocytes.     

Successful separation of apoptosis and necrosis pathways in HaCaT keratinocyte cells induced by UVB irradiation

UVB irradiation can induce apoptotic, necrotic, and differentiation pathways in normal human keratinocytes. The present study was undertaken to determine at what dose of UVB each of these pathways is induced and whether these pathways are distinct or overlapping. We have observed that UVB induces fragmentation of DNA in human HaCaT keratinocytes, in a bimodal manner. Low doses of UVB, 5–20 mJ/cm², increase the levels of apoptosis as shown by increased levels of fragmented DNA, Fas, PARP, and FasL protein, and the number of apoptotic cells as assessed by FACS analysis. At higher doses of UVB (20 and 30 mJ/cm²) the number of apoptotic cells becomes reduced, as does the amount of Fas, PARP, and FasL protein. At these higher doses, cell viability is decreased as measured by DNA synthesis (BrdU labeling) neutral red uptake, which represents an increasing necrotic phenotype. Expression of markers of keratinocyte differentiation, involucrin, keratin K1, and keratin K10, are also observed to decrease with increasing UVB dose. These changes are accompanied by a further increase in DNA fragmentation. We conclude that low doses of UVB (5–20 mJ/cm²) induced an apoptotic pathway, whereas increasing doses (greater than 20 mJ/cm²) of UVB produce a direct necrotic effect and inhibit terminal differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Up-regulation of hyaluronan receptors in Toxoplasma gondii-infected monocytic cells

The apicomplexan, obligate intracellular parasite Toxoplasma gondii orally infects humans and animals. The parasites cross the intestinal epithelium, invade leukocytes in the general circulation and then disseminate into the peripheral organs. The mechanism of extravasation of the infected leukocytes, however, remains poorly understood. It is known that adhesion of leukocytes to extracellular matrix (ECM) is an important factor in extravasation, and CD44 and ICAM-1 on the leukocyte surface are known receptors for hyaluronan (HA), an ECM component. In this study, we demonstrated up-regulation of CD44 and ICAM-1 expression on the surface of T. gondii-infected human monocytic THP-1 cells and fresh isolated human monocyte. T. gondii-infected THP-1 cells adhered more efficiently to immobilized HA than did non-infected cells. T. gondii-infected monocytes in the general circulation might preferentially adhere to the ECM and migrate out from blood vessels, so transporting parasites into the peripheral organs.