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1.
Immunol Lett ; 185: 1-11, 2017 05.
Article in English | MEDLINE | ID: mdl-28274793

ABSTRACT

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies that can result in damage to multiple organs. It is well documented that B cells play a critical role in the development of the disease. We previously showed that protein kinase C associated kinase (PKK) is required for B1 cell development as well as for the survival of recirculating mature B cells and B-lymphoma cells. Here, we investigated the role of PKK in lupus development in a lupus mouse model. We demonstrate that the conditional deletion of PKK in B cells prevents lupus development in Sle1Sle3 mice. The loss of PKK in Sle mice resulted in the amelioration of multiple classical lupus-associated phenotypes and histologic features of lupus nephritis, including marked reduction in the levels of serum autoantibodies, proteinuria, spleen size, peritoneal B-1 cell population and the number of activated CD4 T cells. In addition, the abundance of autoreactive plasma cells normally seen in Sle lupus mice was also significantly decreased in the PKK-deficient Sle mice. Sle B cells deficient in PKK display defective proliferation responses to BCR and LPS stimulation. Consistently, B cell receptor-mediated NF-κB activation, which is required for the survival of activated B cells, was impaired in the PKK-deficient B cells. Taken together, our work uncovers a critical role of PKK in lupus development and suggests that targeting the PKK-mediated pathway may represent a promising therapeutic strategy for lupus treatment.


Subject(s)
B-Lymphocytes/physiology , Lupus Erythematosus, Systemic/immunology , Lupus Nephritis/immunology , Protein Serine-Threonine Kinases/metabolism , Th1 Cells/immunology , Animals , Autoantibodies/metabolism , Cell Proliferation , Cells, Cultured , Disease Models, Animal , Female , Humans , Lipopolysaccharides/immunology , Lymphocyte Activation/genetics , Mice , Mice, Knockout , Molecular Targeted Therapy , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/genetics
2.
Methods Inf Med ; 47(1): 58-62, 2008.
Article in English | MEDLINE | ID: mdl-18213429

ABSTRACT

OBJECTIVE: To understand whether home health technology in the market and in development can satisfy the needs of patients and their non-professional caregivers for proactive support in managing health and chronic conditions in the home. METHODS: A panel of clinical providers and technology researchers was assembled to examine whether home health technology addresses consumer-defined requirements for self-care devices. A lexicon of home care and self-care technology terms was then created. A global survey of home health technology for patients with heart disease and dementia was conducted. The 254 items identified were categorized by conditions treated, primary user, function, and purpose. A focus group of patients and caregivers was convened to describe their expectations of self-care technology. Items identified in the database were then assessed for these attributes. RESULTS: Patients and family caregivers indicated a need for intelligent self-care technology which supports early diagnosis of health changes, intervention enablement, and improvement of communication quality among patients and the health care system. Of these, only intervention enablement was commonly found in the home health technology items identified. CONCLUSIONS: An opportunity exists to meet consumer self-care needs through increased research and development in intelligent self-care technology.


Subject(s)
Activities of Daily Living , Artificial Intelligence , Home Care Services/organization & administration , Personal Health Services , Self Care/instrumentation , Self-Help Devices , Caregivers , Chronic Disease , Focus Groups , Health Care Surveys , Humans , Patient Care Team , Patient Satisfaction , Self Care/methods
3.
Br J Cancer ; 97(1): 14-21, 2007 Jul 02.
Article in English | MEDLINE | ID: mdl-17565347

ABSTRACT

Cancer patients may experience skin problems while undergoing chemotherapy and radiation therapy. Frequency of skin reactions may be influenced by skin pigmentation and psychological factors. A Symptom Inventory completed by 656 cancer patients nationwide before and after chemotherapy, radiation therapy, or chemotherapy plus radiation therapy was analysed to determine if treatment type, race (Black vs White), and pretreatment expectations influenced post-treatment skin reactions. Subsequent analysis of a local Symptom Inventory completed weekly for 5 weeks by 308 patients receiving radiation therapy examined severity of reported skin reactions. Significantly more patients receiving radiation therapy had stronger expectations of skin problems (62%) than patients receiving chemotherapy (40%, P=0.001) or chemotherapy plus radiation therapy (45%, P=0.003). Overall, expectations did not correlate with patient reported post-treatment skin problems in white (r=0.014, P=0.781) or black (r=0.021, P=0.936) patients. Although no significant difference was found between black and white patients in their pretreatment expectations of skin problems (P=0.32), black patients (10 out of 18, 56%) reported more skin problems than white patients (90 out of 393, 23%, P=0.001). Similarly, the local study showed that significantly more black patients (1 out of 5, 20%) reported severe skin reactions at the treatment site than white patients (12 out of 161, 8%). A direct correlation was observed between severity of skin problems and pain at the treatment site (r=0.541, P<0.001). Total radiation exposure did not significantly correlate with the report of skin problems at the treatment site for white or black patients. Overall, black patients reported more severe post-treatment skin problems than white patients. Our results suggest that symptom management for post-treatment skin reactions in cancer patients receiving radiation treatment could differ depending on their racial background.


Subject(s)
Antineoplastic Agents/adverse effects , Black or African American , Neoplasms/drug therapy , Neoplasms/radiotherapy , Radiotherapy/adverse effects , Skin Diseases/epidemiology , White People , Drug Eruptions/epidemiology , Female , Humans , Male , Middle Aged , Pain/psychology , Radiodermatitis/epidemiology , Skin Diseases/chemically induced , Surveys and Questionnaires
4.
Am J Physiol Cell Physiol ; 278(4): C822-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10751330

ABSTRACT

PGE(2) levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE(2) production in activated keratinocytes are unclear. In previous studies, we showed that PGE(2) is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE(2) synthesis is an important component of wound healing. Here, we examine the role of phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) enzymes in modulation of PGE(2) production. We report that the increased PGE(2) production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA(2) (sPLA(2)) proteins. Increased sPLA(2) activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA(2), along with the M-type sPLA(2) receptor. Thus the coordinate expression of sPLA(2) and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair.


Subject(s)
Isoenzymes/metabolism , Keratinocytes/physiology , Phospholipases A/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Cells, Cultured , Cyclooxygenase 2 , Dinoprostone/metabolism , Fatty Acids/metabolism , Humans , Keratinocytes/enzymology , Keratinocytes/metabolism , Keratinocytes/pathology , Membrane Proteins , Receptors, Cell Surface/metabolism , Receptors, Phospholipase A2 , Reverse Transcriptase Polymerase Chain Reaction , Wound Healing/physiology
5.
Carcinogenesis ; 20(10): 1939-44, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10506108

ABSTRACT

UV light is a complete carcinogen, inducing both basal and squamous cell skin cancers. The work described uses the selective COX-2 inhibitor celecoxib to examine the efficacy of COX-2 inhibition in the reduction of UV light-induced skin tumor formation in hairless mice. UVA-340 sun lamps were chosen as a light source that effectively mimics the solar UVA and UVB spectrum. Hairless mice were irradiated for 5 days a week for a total dose of 2.62 J/cm(2). When 90% of the animals had at least one tumor, the mice were divided into two groups so that the tumor number and multiplicity were the same (P < 0.31). Half of the mice were then fed a diet containing 1500 p.p.m. celecoxib. Tumor number, multiplicity and size were then observed for the next 10 weeks. Ninety-five percent of the tumors formed were histopathologically evaluated as squamous cell carcinoma. COX-2 expression and activity were increased in tumors. After 10 weeks, the difference in tumor number and multiplicity in the drug-treated group was 56% of UV controls (P < 0.001). The results show that the orally administered selective COX-2 inhibitor celecoxib prevents new tumor formation after the onset of photocarcinogenesis and suggest that treatment with celecoxib may be very useful in preventing UV-induced skin tumors in humans.


Subject(s)
Carcinoma, Squamous Cell/prevention & control , Cyclooxygenase Inhibitors/therapeutic use , Isoenzymes/drug effects , Neoplasms, Radiation-Induced/prevention & control , Prostaglandin-Endoperoxide Synthases/drug effects , Skin Neoplasms/prevention & control , Sulfonamides/therapeutic use , Animals , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/pathology , Celecoxib , Cell Division , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Female , Immunohistochemistry , Isoenzymes/metabolism , Mice , Mice, Hairless , Neoplasms, Radiation-Induced/enzymology , Neoplasms, Radiation-Induced/pathology , Prostaglandin-Endoperoxide Synthases/metabolism , Pyrazoles , Skin Neoplasms/enzymology , Skin Neoplasms/pathology , Ultraviolet Rays
7.
Carcinogenesis ; 19(5): 723-9, 1998 May.
Article in English | MEDLINE | ID: mdl-9635856

ABSTRACT

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.


Subject(s)
Isoenzymes/genetics , Neoplasms, Radiation-Induced/enzymology , Prostaglandin-Endoperoxide Synthases/genetics , Skin Neoplasms/enzymology , Skin/radiation effects , Carcinoma, Basal Cell/enzymology , Carcinoma, Squamous Cell/enzymology , Cells, Cultured , Cyclooxygenase 2 , Enzyme Induction , Gene Expression Regulation, Enzymologic/radiation effects , Humans , Immunohistochemistry , Isoenzymes/biosynthesis , Keratinocytes/enzymology , Keratinocytes/radiation effects , Keratosis/enzymology , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/biosynthesis , Skin/cytology , Skin/enzymology , Skin Neoplasms/etiology , Ultraviolet Rays
8.
J Biol Chem ; 273(21): 12901-8, 1998 May 22.
Article in English | MEDLINE | ID: mdl-9582321

ABSTRACT

The mitogen-activated protein kinase (MAPK) cascade is believed to function as an important regulator of prostaglandin biosynthesis. Previously we reported that interleukin-1beta induces activation of JNK/SAPK and p38 MAPK with concomitant up-regulation of cyclooxygenase (Cox)-2 expression and prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of DeltaMEKK1 (a constitutively active truncation mutant of MEKK1 containing the C-terminal 324 amino acids) increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. DeltaMEKK1 overexpression results in activation of both c-Jun N-terminal kinases/extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore, activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 --> SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2 production. We also found that overexpression of the constitutively active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK phosphorylation, and increases PGE2 production and Cox-2 expression. By comparison, overexpression of the dominant negative form of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a potential role for the MEKK1 --> SEK1/MKK4 --> p38 MAPK -->--> Cox-2 cascade linking members of the MAPK pathway with prostaglandin biosynthesis.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Isoenzymes/biosynthesis , MAP Kinase Kinase 4 , MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Prostaglandin-Endoperoxide Synthases/biosynthesis , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , 3T3 Cells , Animals , Cyclooxygenase 2 , Dinoprostone/biosynthesis , Enzyme Activation , Enzyme Induction , Mice , Phosphorylation , p38 Mitogen-Activated Protein Kinases
9.
Biochim Biophys Acta ; 1401(2): 221-34, 1998 Feb 04.
Article in English | MEDLINE | ID: mdl-9531979

ABSTRACT

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.


Subject(s)
Keratinocytes/cytology , Keratinocytes/physiology , Receptors, Prostaglandin E/physiology , Abortifacient Agents, Nonsteroidal/pharmacology , Adenylyl Cyclases/drug effects , Bromodeoxyuridine/pharmacokinetics , Bucladesine/pharmacology , Cell Division/drug effects , Cell Division/physiology , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/administration & dosage , Dinoprostone/analogs & derivatives , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Gene Expression/genetics , Gene Expression/physiology , Humans , Indomethacin/antagonists & inhibitors , Indomethacin/pharmacology , Keratinocytes/drug effects , Misoprostol/administration & dosage , Misoprostol/pharmacology , Oxytocics/administration & dosage , Oxytocics/pharmacology , RNA/analysis , RNA/genetics , Receptors, Prostaglandin E/genetics , Receptors, Prostaglandin E, EP2 Subtype , Receptors, Prostaglandin E, EP3 Subtype , Transcription, Genetic/genetics , Transcription, Genetic/physiology
10.
Mol Biol Cell ; 8(5): 811-24, 1997 May.
Article in English | MEDLINE | ID: mdl-9168468

ABSTRACT

Calcium concentration influences keratinocyte differentiation, and, following injury, keratinocytes move through an environment of changing calcium levels. Because these migrating cells in wounds invariably express collagenase 1, we assessed if modulation of calcium levels regulates collagenase 1 production by primary human keratinocytes. Accurately reflecting the confined spatial pattern of enzyme production seen in vivo, collagenase 1 mRNA was expressed only by keratinocytes migrating from foci of differentiated cells. Treatment with calcium ionophores A23187 or thapsigargin markedly inhibited the basal and phorbol 12-myristate 13-acetate-(PMA) stimulated accumulation of keratinocyte collagenase 1 in the medium but did not affect collagenase 1 production by control or PMA-treated fibroblasts. A23187-mediated inhibition of collagenase 1 protein was not associated with a decrease in mRNA levels but rather was controlled by a selective and reversible block of enzyme secretion. This block in secretion was likely not due to altered protein folding as the proenzyme within A23187-treated cells remained capable of autolytic activation upon treatment with p-aminophenylmercuric acetate. In contrast, 92-kDa gelatinase mRNA and secreted protein levels were coordinately reduced by A23187. Keratin 14 expression, a basal keratinocyte marker, was reduced with PMA treatment, but A23187 did not affect keratin 14 expression. In human wounds, both basal and suprabasal keratinocytes at the migrating front of epidermis stained for keratin 14, but only the basal cells expressed collagenase 1. These data suggest that collagenase 1 production is not necessarily linked with expression of basal cell markers and that modulation of intracellular calcium levels can block secretion of collagenase 1 by keratinocytes which have moved away from the stratum basalis and from their natural substrate.


Subject(s)
Calcium/metabolism , Collagenases/metabolism , Keratinocytes/enzymology , Calcimycin/pharmacology , Cell Differentiation , Cell Movement , Cells, Cultured , Collagenases/genetics , Humans , Ionophores/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Matrix Metalloproteinase 1 , Matrix Metalloproteinase Inhibitors , RNA, Messenger/metabolism , Thapsigargin
11.
Am J Physiol ; 270(4 Pt 1): C1037-50, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8928731

ABSTRACT

Ultraviolet light (UV) B-induced inflammation is characterized by dramatic increases in prostaglandin E2 (PGE2) synthesis due to enhanced arachidonate deacylation from the membrane. Therefore, the effect of UV on sythesis, mass, and distribution of the high-molecular-weight phospholipase A2 (cPLA2) in cultured human keratinocytes and human skin was studied. The 105-kDa cPLA2 was demonstrated to be the critical enzyme in UV-induced PGE2 synthesis and erythema in the first 6 h postirradiation. Immunoprecipitation of 35S-labeled protein showed cPLA2 synthesis increased three- to fourfold 6 h after irradiation. Immunoprecipitated 32P-labeled cPLA2 demonstrated phosphorylation of cPLA2 was concurrently induced, suggesting that UV also activates cPLA2. This increase in cPLA2 synthesis and activation also closely correlated with increased PGE2 synthesis and [3H]arachidonic acid release and was effectively blocked by both an S-oligonucleotide antisense to cPLA2 and methyl arachidonate fluorophosphate, a specific inhibitor of cPLA2. Biopsy and histochemical examination of erythematous sites expressed increased amounts of cPLA2 whereas nonerythematous irradiated sites did not. In contrast, cyclooxygenase-1 and -2 in cultures and skin explants were unaffected 6 h post-UV, and no change in cyclooxygenase activity was observed at this time. These results suggest that increased cPLA2 synthesis occurs only when skin is exposed to UV doses that are sufficient to cause erythema and indicate expression of cPLA2 participates in acute UV inflammation.


Subject(s)
Cytosol/enzymology , Dinoprostone/biosynthesis , Phospholipases A/biosynthesis , Skin/metabolism , Skin/radiation effects , Ultraviolet Rays , Amino Acid Sequence , Animals , Arachidonic Acid/metabolism , Dose-Response Relationship, Radiation , Erythema/enzymology , Erythema/etiology , Humans , Keratinocytes/metabolism , Molecular Sequence Data , Molecular Weight , Phospholipases A/chemistry , Phospholipases A2 , Radiation Injuries, Experimental/enzymology , Skin/cytology , Time Factors
13.
J Invest Dermatol ; 106(4): 785-9, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8618023

ABSTRACT

Human epidermal cell cultures were examined to determine whether they were capable of histamine release. Results of these studies indicated that keratinocytes contain and release significant amounts of histamine. In the skin of some individuals, histamine content was induced after ultraviolet B light injury, and 40% of subjects demonstrated high basal histamine levels. Mass spectrometric analysis of cell supernatants showed that the histamine was released into the extracellular environment. Such release may contribute to common itching or intensify the inflammatory response in vivo.


Subject(s)
Histamine/analysis , Keratinocytes/chemistry , Keratinocytes/radiation effects , Ultraviolet Rays/adverse effects , Adult , Cells, Cultured , Dermatitis/etiology , Histamine Release/radiation effects , Humans , Keratinocytes/metabolism
14.
Biochim Biophys Acta ; 1299(1): 23-33, 1996 Jan 05.
Article in English | MEDLINE | ID: mdl-8555249

ABSTRACT

UVB irradiation has previously been shown to significantly increase phospholipase activity and prostaglandin synthesis. Because UVB irradiation is a potent oxidative stress, the role of active oxygen species in regulating UV-induced cPLA2 synthesis and phosphorylation was examined. In the present study, irradiation produced a 3-fold increase in synthesis within 6 h following irradiation. Phosphorylation of cPLA2 was also increased to a similar extent. UVB-induced synthesis and phosphorylation of cPLA2 could be inhibited by pretreatment with the antioxidants 2,2,5,7,8-pentamethyl-6-hydroxychromane (50 microM) or N-acetylcysteine (10 mM). Treatment of unirradiated cultures with the potent oxidant tert-butyl hydroperoxide (500 microM) also increased cPLA2 synthesis and phosphorylation, suggesting that oxidative injury is an important regulator of cPLA2 synthesis. Increased synthesis of cPLA2 correlated well with increased [3H]arachidonic acid release, PGE2 synthesis and lipid peroxidation in epidermis after oxidant or UVB treatment. The results indicate that UVB-induced upregulation of cPLA2 synthesis is mediated by UVB-induced formation of free radicals.


Subject(s)
Oxidative Stress , Phospholipases A/biosynthesis , Ultraviolet Rays , Acetylcysteine/pharmacology , Antioxidants/pharmacology , Arachidonic Acid/metabolism , Cells, Cultured/drug effects , Cells, Cultured/radiation effects , Cytosol/enzymology , Dinoprostone/biosynthesis , Enzyme Induction/drug effects , Enzyme Induction/radiation effects , Humans , Lipid Peroxidation , Peroxides/pharmacology , Phospholipases A2 , Phosphorylation , Skin/drug effects , Skin/radiation effects , tert-Butylhydroperoxide
15.
Invest Ophthalmol Vis Sci ; 37(1): 142-53, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8550317

ABSTRACT

PURPOSE: To study the functional significance of prostaglandin synthesis after ultraviolet-B (UVB) exposure of cultured human lens epithelial cells and rabbit eyes in vivo. METHODS: Prostaglandin E2 (PGE2) was assayed using a radioimmunoassay (RIA) and mass spectroscopy. An immortalized human lens epithelial cell line (HLE-B3) was exposed to UV irradiation, and the synthesis of PGE2 was compared with the rabbit lens epithelial cell line N/N1003A. Intact human lenses were exposed to UVB in organ culture. [3H]Thymidine incorporation was measured in cultured lens epithelial cells by incubation with the radiolabel. The effects of isobutyl methyl xanthine (IBMX), an inhibitor of phosphodiesterase and of dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, on PGE2 synthesis and DNA synthesis, were determined. Rabbit eyes were exposed to UVB radiation in vivo. Intraocular pressure was measured at specific times after exposure. Aqueous humor was remove from rabbit eyes, and its PGE2 content was measured by RIA. RESULTS: Cultured human lens epithelial cells (HLE), like rabbit lens epithelial cells (RLE), showed a dose-dependent increase in basal PGE2 synthesis 24 hours after UVB exposure. However, the amount of PGE2 synthesis was 2000-fold higher in the rabbit cells. Ultraviolet-B radiation enhanced the incorporation of [3H]thymidine in lens epithelial cells. Pretreatment of cells with indomethacin reduce PGE2 synthesis and [3H]thymidine incorporation. The human and rabbit cells responded in a similar manner to changes in DNA synthesis after UVB exposure. The addition of IBMX or dbcAMP to indomethacin-treated, UVB-exposed cells restored DNA synthesis toward the levels observed in the UVB-exposed cells. An increase in the concentration of cAMP was observed in lens epithelial cells exposed to exogenous PGE2. PGE2 synthesis in intact human lenses also increased twofold 24 hours after UVB exposure. Exposure of the rabbit eye in vivo to an optimal dose of UVB produced an increase in the PGE2 levels of the lens and the aqueous humor. Measurements of the intraocular pressure (IOP) of the animals showed a decrease in IOP by 2.21 +/- 0.66 and 6.45 +/- 0.79 mm Hg (mean +/- SEM, P = 0.004, t-test) at 6 and 24 hours after UVB exposure, respectively. The decrease in IOP was prevented by pretreatment with indomethacin. Exposure of the rabbit lens to UVB radiation in vivo enhanced [3H]thymidine incorporation twofold into the lens. Pretreatment of rabbits with indomethacin before exposure reduced this response. CONCLUSIONS: Results indicate that UVB exposure enhances PGE2 synthesis in HLE cultures as well as in rabbit lenses irradiated in vivo. This increased PGE2 synthesis is related to the increase in DNA synthesis observed after UVB treatment. The modulation of DNA synthesis in cultured lens epithelial cells after UVB exposure may be mediated by a cAMP-dependent mechanism.


Subject(s)
DNA/biosynthesis , Dinoprostone/biosynthesis , Intraocular Pressure/radiation effects , Lens, Crystalline/radiation effects , Ultraviolet Rays , 1-Methyl-3-isobutylxanthine/pharmacology , Animals , Autoradiography , Cell Division/radiation effects , Cell Line , Cells, Cultured , Cyclic AMP/metabolism , Cyclooxygenase Inhibitors/pharmacology , DNA/radiation effects , DNA Replication/radiation effects , Dinoprostone/radiation effects , Dose-Response Relationship, Radiation , Epithelium/drug effects , Epithelium/radiation effects , Humans , Indomethacin/pharmacology , Infant , Lens, Crystalline/cytology , Lens, Crystalline/drug effects , Mass Spectrometry , Ocular Hypertension/etiology , Phosphodiesterase Inhibitors/pharmacology , Rabbits , Radioimmunoassay
16.
J Invest Dermatol ; 104(1): 52-7, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7798641

ABSTRACT

The regulatory effect of endogenously synthesized eicosanoid metabolites on the expression of tissue inhibitor of metalloproteinases (TIMP), interstitial collagenase, and 92-kDa gelatinase by human macrophages was examined. TIMP and metalloproteinase production were stimulated with three agonists that produce distinct patterns of eicosanoid synthesis: lipopolysaccharide (10 micrograms/ml), denatured collagen (10 micrograms/ml), or zymosan (1 mg/ml). Indomethacin (3 micrograms/ml) or MK886 (3 microM), a specific inhibitor of 5-lipoxygenase, was used to examine the role of endogenous metabolites of arachidonic acid. Regardless of the agonist used, TIMP production by macrophages was inhibited 65% by indomethacin, synthesis of interstitial collagenase was reduced 70%, and expression of 92-kDa gelatinase was decreased 40%. In contrast, inhibition of leukotriene synthesis had no effect on metalloproteinase or TIMP production. The agonist-stimulated increase in TIMP and collagenase production was directly correlated to the cumulative prostaglandin E2 level induced by the agonist used. However, if response to an agonist was poor, the exogenous addition of prostaglandin E2 could not increase TIMP or collagenase production more than twofold, indicating an important permissive effect of the agonist on the regulation of each protein's expression. The mechanism of indomethacin inhibition of TIMP and collagenase production was studied by labeling the cells with [35S]-methionine and performing immunoprecipitation using specific antiserum. Indomethacin markedly inhibited the lipopolysaccharide-induced biosynthesis of both TIMP and collagenase. Northern analysis revealed parallel suppression of TIMP and collagenase steady-state mRNA levels by indomethacin, indicating pretranslational control. The regulation of inflammatory-cell TIMP and interstitial collagenase expression by prostaglandin E2 suggests that therapy inhibiting the cellular response to prostaglandins may be useful in cutaneous and systemic disease states involving macrophage-mediated connective-tissue destruction.


Subject(s)
Dinoprostone/biosynthesis , Glycoproteins/agonists , Macrophages, Alveolar/metabolism , Adult , Collagenases/biosynthesis , Collagenases/metabolism , Dinoprostone/pharmacology , Glycoproteins/biosynthesis , Humans , Precipitin Tests , Protein Processing, Post-Translational , Tissue Inhibitor of Metalloproteinases
17.
J Biol Chem ; 269(47): 30022-9, 1994 Nov 25.
Article in English | MEDLINE | ID: mdl-7962003

ABSTRACT

We have previously shown that during wound healing migrating keratinocytes, which are in contact with the dermal matrix, express interstitial collagenase, whereas basal epidermal cells, which reside on an intact basement membrane, do not. Duplicating this in vivo pattern, collagenase production was induced in primary human keratinocytes grown on native type I collagen, but only background levels of enzyme were detected in cells cultured on denatured type I collagen or on Matrigel. Using genistein, herbimycin A, and sodium orthovanadate, we show that tyrosine kinase activity was required for collagen-mediated induction of keratinocyte collagenase. Similarly, collagenase steady-state mRNA levels and the activity of a transfected human collagenase-promoter CAT construct were inhibited by genistein and enhanced by orthovanadate. Staurosporine and H-7 also blocked collagenase production, indicating that protein kinase C activity was also required for collagen-mediated induction of keratinocyte collagenase. All inhibitory effects were dose-dependent, and no compound significantly affected total protein synthesis. Furthermore, both tyrosine kinase and protein kinase C inhibitors blocked phorbol ester-mediated induction of collagenase, but only protein kinase C antagonists abrogated phorbol ester-mediated induction of c-fos mRNA. These data suggest that similar signal transduction pathways are used by various agonists to mediate the stimulation of interstitial collagenase production.


Subject(s)
Collagen/pharmacology , Collagenases/biosynthesis , Keratinocytes/drug effects , Protein Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Alkaloids/pharmacology , Cells, Cultured , Collagenases/genetics , Enzyme Induction , GTP-Binding Proteins/metabolism , Genes, fos , Genistein , Humans , Isoflavones/pharmacology , Keratinocytes/enzymology , Prostaglandins/metabolism , Protein Kinase Inhibitors , RNA, Messenger/genetics , RNA, Messenger/metabolism , Staurosporine , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic/drug effects
18.
J Clin Invest ; 94(3): 965-77, 1994 Sep.
Article in English | MEDLINE | ID: mdl-8083381

ABSTRACT

The pathogenic gram-positive bacterium Streptococcus pyogenes (group A streptococcus) causes numerous diseases of cutaneous tissue, each of which is initiated after the interaction of the bacterium with the cells of the epidermis. In this study, we show that different surface proteins of S. pyogenes play important roles in determining the cell-specific tropism of the bacterium in skin. Using streptococcal strains with defined mutations in the genes which encode surface proteins in combination with primary cultures of human skin and an in situ adherence assay which uses histological sections of human skin, we show that the M protein of S. pyogenes mediates the binding of the bacterium to keratinocytes, while a second streptococcal surface protein, protein F, directs the adherence of the organism to Langerhans' cells. Characterization of binding revealed that adherence was inhibited by purified streptococcal proteins and pretreatment of both host cells with the protease trypsin. Adherence was only slightly affected by the state of keratinocyte differentiation in vitro, but was considerably modulated in response to environmental conditions known to regulate expression of M protein and protein F, suggesting that the interaction between these bacterial cell-surface structures/adhesins and keratinocytes and Langerhans' cells may play an important role in streptococcal skin disease.


Subject(s)
Antigens, Bacterial , Bacterial Adhesion , Bacterial Outer Membrane Proteins , Bacterial Proteins/physiology , Carrier Proteins , Porins/metabolism , Skin/microbiology , Streptococcus pyogenes/pathogenicity , Bacterial Adhesion/drug effects , Cells, Cultured , Epidermis/microbiology , Humans , In Vitro Techniques , Langerhans Cells/microbiology , Melanocytes/microbiology , Streptococcus pyogenes/physiology , Trypsin/pharmacology
19.
J Clin Invest ; 94(1): 79-88, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8040294

ABSTRACT

Wound repair involves cell migration and tissue remodeling, and these ordered and regulated processes are facilitated by matrix-degrading proteases. We reported that interstitial collagenase is invariantly expressed by basal keratinocytes at the migrating front of healing epidermis (Saarialho-Kere, U. K., E. S. Chang, H. G. Welgus, and W. C. Parks, 1992. J. Clin. Invest. 90:1952-1957). Because of the limited substrate specificity of collagenase, principally for interstitial fibrillar collagens, other enzymes must also be produced in the wound environment to effectively restructure tissues with a complex matrix composition. Stromelysins-1 and -2 are closely related, yet distinct metalloproteinases, and both can degrade many noncollagenous connective tissue macromolecules. Using in situ hybridization and immunohistochemistry, we found that both stromelysins are produced by distinct populations of keratinocytes in a variety of chronic ulcers. Stromelysin-1 mRNA and protein were detected in basal keratinocytes adjacent to but distal from the wound edge in what probably represents the sites of proliferating epidermis. In contrast, stromelysin-2 mRNA was seen only in basal keratinocytes at the migrating front, in the same epidermal cell population that expresses collagenase. Stromelysin-1-producing keratinocytes resided on the basement membrane, whereas stromelysin-2-producing keratinocytes were in contact with the dermal matrix. Furthermore, stromelysin-1 expression was prominent in dermal fibroblasts, whereas no signal for stromelysin-2 was seen in any dermal cell. These findings demonstrate that stromelysins-1 and -2 are produced by different populations of basal keratinocytes in response to wounding and suggest that these two matrix metalloproteinases serve distinct roles in tissue repair.


Subject(s)
Keratinocytes/metabolism , Metalloendopeptidases/biosynthesis , Wound Healing , Cells, Cultured , Chronic Disease , Collagenases/biosynthesis , Collagenases/genetics , Fibroblasts/metabolism , Humans , Matrix Metalloproteinase 10 , Matrix Metalloproteinase 3 , Metalloendopeptidases/genetics , RNA, Messenger/analysis , Ulcer/metabolism
20.
J Biol Chem ; 269(5): 3529-33, 1994 Feb 04.
Article in English | MEDLINE | ID: mdl-8106395

ABSTRACT

To study the signal transduction mechanisms by which ultraviolet B (UVB) leads to increased prostaglandin E2 (PGE2) synthesis, human epidermal cultures were irradiated with 30 mJ/cm2 UVB and assayed for 6-h cumulative PGE2. Supernatants from irradiated cultures showed a 4-fold increase in PGE2 synthesis (113.6 +/- 26.8 pg/mg protein) when compared to supernatants from sham-irradiated cultures (25.6 +/- 3.9 pg/mg protein). Pretreatment of irradiated cultures with genistein (10 micrograms/ml) or tyrphostin-23 (50 microM), inhibitors of tyrosine kinases, blocked UVB-stimulated PGE2 synthesis. Treatment of nonirradiated cultures with epidermal growth factor (EGF), which acts through the receptor tyrosine kinase EGF-R, produced a 4-fold increase in PGE2 synthesis. However, addition of EGF to irradiated cultures did not further enhance their PGE2 synthesis, indicating irradiation rendered them refractory to EGF stimulation. In contrast, irradiated cultures could still significantly increase their PGE2 synthesis in response to the calcium ionophore A23187 or the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate, suggesting that the lack of response to EGF was selective. Furthermore, anti-phosphotyrosine immunoblot analysis revealed UVB-induced phosphorylation of tyrosine residues of EGF-R, an indicator of receptor activation. Phosphorylation was maximal 30-60 min after irradiation and was blocked by the tyrosine kinase inhibitors, genistein and tyrphostin. The antioxidant N-acetylcysteine decreased UVB-induced EGF-R tyrosine phosphorylation and PGE2 synthesis to near-basal levels. Conversely, treatment of unirradiated cultures with the potent oxidant tert-butyl-hydroperoxide (100 microM) increased both PGE2 synthesis and EGF-R phosphorylation. Collectively, these data suggest that antioxidant depletion induced by UV results in tyrosine phosphorylation and activation of the EGF-R. This activation may subsequently activate epidermal phospholipase at early time points after UVB exposure.


Subject(s)
Dinoprostone/biosynthesis , ErbB Receptors/metabolism , Keratinocytes/radiation effects , Protein-Tyrosine Kinases/metabolism , Signal Transduction/radiation effects , Skin/radiation effects , Tyrphostins , Ultraviolet Rays , Calcimycin/pharmacology , Catechols/pharmacology , Cells, Cultured , Enzyme Activation , Epidermal Growth Factor/pharmacology , ErbB Receptors/radiation effects , Female , Genistein , Humans , Isoflavones/pharmacology , Keratinocytes/enzymology , Keratinocytes/metabolism , Kinetics , Nitriles/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Skin/enzymology , Skin/metabolism , Tetradecanoylphorbol Acetate/pharmacology
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