Hurpin is a selective inhibitor of lysosomal cathepsin L and protects keratinocytes from ultraviolet-induced apoptosis

Hurpin (headpin/PI13/serpinB13) is an intracellular, differentially spliced member of the serpin superfamily that has been linked to differentiation and apoptosis of human keratinocytes. It is transiently downregulated by UV light and overexpressed in psoriatic skin lesions. Although it has all of the features of an inhibitory serpin, a productive interaction between hurpin and a proteinase has not yet been reported. Here we demonstrate that hurpin is a potent and selective inhibitor of the archetypal lysosomal cysteine proteinase cathepsin L (catL). Recombinant hurpin inhibits human catL with a stoichiometry of inhibition (SI) of 1.7 and a rate constant k(assoc) of (4.6 +/- 0.14) x 10(5) M(-1) s(-1). It inefficiently inhibits catV and does not inhibit papain, catB, or catK. To investigate the inhibitory mechanism, we determined the P1-P1' bond in the reactive center loop cleaved by catL ((356)Thr-(357)Ser) and expressed variants in which the proximal hinge, P1 residue, or differentially spliced CD loop was mutated. The results of assays using these proteins suggest that inhibition of catL by hurpin occurs via the conventional serpin inhibitory mechanism and that the CD loop plays no role in the process. Finally, it was found that the majority of hurpin is cytosolic and that its overexpression in human keratinocytes confers resistance to UV-induced apoptosis. Given that lysosomal disruption, release of catL, and catL-mediated caspase activation are known to occur in response to cellular stress, we propose that a physiological role of hurpin is to protect epithelial cells from ectopic catL.     

Inhibition of human kallikreins 5 and 7 by the serine protease inhibitor lympho-epithelial Kazal-type inhibitor (LEKTI)

LEKTI is a 120-kDa protein that plays an important role in skin development, as mutations affecting LEKTI synthesis underlie Netherton syndrome, an inherited skin disorder producing severe scaling. Its primary sequence indicates that the protein consists of 15 domains, all resembling a Kazal-type serine protease inhibitor. LEKTI and two serine proteases belonging to the human tissue kallikrein (hK) family (hK5 and hK7) are expressed in the granular layer of skin. In this study, we characterize the interaction of two recombinant LEKTI fragments containing three or four intact Kazal domains (domains 6-8 and 9-12) with recombinant rhK5, a trypsin-like protease, and recombinant rhK7, a chymotrypsin-like protease. Both fragments inhibited rhK5 similarly in binding and kinetic studies performed at pH 8.0, as well as pH 5.0, the pH of the stratum corneum where both LEKTI and proteases may function. Inhibition equilibrium constants (Ki) measured either directly in concentration-dependent studies or calculated from measured association (kass) and dissociation (kdis) rate constants were 1.2-5.5 nM at pH 8.0 and 10-20 nM at pH 5.0. At pH 8.0, kass and kdis values were 4.7 x 10(5) M(-1) s(-1) and 5.5 x 10(-4) s(-1), and at pH 5.0 they were 4.0 x 10(4) M(-1) s(-1) and 4.3 x 10(-4) s(-1), respectively. The low Ki and kdis values (t1/2 of 20-25 min) indicate tight and specific association. Only fragment 6-9' was a good inhibitor of rhK7, demonstrating a Ki of 11 nM at pH 8.0 in a reaction that was rapidly reversible. These results show that LEKTI, at least in fragment form, is a potent inhibitor of rhK5 and that this protease may be a target of LEKTI in human skin.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

Targeted disruption of SPI3/Serpinb6 does not result in developmental or growth defects, leukocyte dysfunction, or susceptibility to stroke

Protease inhibitor 6 (PI-6/SERPINB6) is a widely expressed nucleocytoplasmic serpin. It inhibits granulocyte cathepsin G and neuronal neuropsin, and it is thought to protect cells from death caused by ectopic release or internalization of protease during stress such as infection or cerebral ischemia. To probe the biological functions of PI-6, we generated mice lacking its ortholog (SPI3/Serpinb6). SPI3-deficient mice developed normally and were fertile, and no abnormal pathology or increased sensitivity to cerebral ischemia was observed. There were no perturbations in leukocyte development or numbers, and recruitment of leukocytes to the peritoneal cavity was normal. SPI3-deficient mice were equally susceptible as wild-type mice to systemic Candida albicans infection, although there was a slight decrease in the ability of neutrophils from SPI3-deficient mice to kill C. albicans in vitro. Increased levels of a related inhibitor Serpinb1 (monocyte/neutrophil elastase inhibitor) in the tissues of targeted mice suggests that compensation by other serpins reduces the impact of SPI3 deficiency in these animals and may explain the lack of a more obvious phenotype.     

Intrahepatic lymphocyte expression of dipeptidyl peptidase I-processed granzyme B and perforin induces hepatocyte expression of serine proteinase inhibitor 6 (Serpinb9/SPI-6)

Human proteinase inhibitor 9 (PI-9/serpinB9) and the murine ortholog, serine proteinase inhibitor 6 (SPI-6/serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, APCs, and CTLs protects these cells against the actions of granzyme B, and when expressed in tumor cells or virally infected hepatocytes, confers resistance to killing by CTL and NK cells. The present studies were designed to assess the existence of any correlation between granzyme B activity in intrahepatic lymphocytes and induction of hepatic SPI-6 expression. To this end, SPI-6, PI-9, and serpinB9 homolog expression was examined in response to IFN-alpha treatment and during in vivo adenoviral infection of the liver. SPI-6 mRNA expression increased 10- to 100-fold in the liver after IFN-alpha stimulation and during the course of viral infection, whereas no significant up-regulation of SPI-8 and <5-fold increases in other PI-9/serpinB9 homolog mRNAs was observed. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL. Moreover, IFN-alpha-induced up-regulation of hepatocyte SPI-6 mRNA expression was not observed in NK cell-depleted mice. Additional experiments using genetically altered mice either deficient in perforin or unable to process or express granzyme B indicated that SPI-6 is selectively up-regulated in hepatocytes in response to infiltration of the liver by NK cells that express perforin and enzymatically active granzyme B.     

SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation

SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS.     

DNase 2 is the main DNA-degrading enzyme of the stratum corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA.     

Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha. Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells with myo-[(3)H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells. The addition of PI-TPalpha to a total lysate of myo-[(3)H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca(2+) further increased this formation. Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.     

Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression

Human kallikrein 5 (KLK5) is a member of the human kallikrein gene family of serine proteases. Preliminary results indicate that the protein, hK5, may be a potential serological marker for breast and ovarian cancer. Other studies implicate hK5 with skin desquamation and skin diseases. To gain further insights on hK5 physiological functions, we studied its substrate specificity, the regulation of its activity by various inhibitors, and identified candidate physiological substrates. After producing and purifying recombinant hK5 in yeast, we determined the k(cat)/K(m) ratio of the fluorogenic substrates Gly-Pro-Arg-AMC and Gly-Pro-Lys-AMC, and showed that it has trypsin-like activity with strong preference for Arg over Lys in the P1 position. The serpins alpha(2)-antiplasmin and antithrombin were able to inhibit hK5 with an inhibition constant (k(+2)/K(i)) of 1.0 x 10(-) (2)and 4.2 x 10(-4) m(-1) min(-1), respectively. No inhibition was observed with the serpins alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, although alpha(2)-macroglobulin partially inhibited hK5 at high concentrations. We also demonstrated that hK5 can efficiently digest the extracellular matrix components, collagens type I, II, III, and IV, fibronectin, and laminin. Furthermore, our results suggest that hK5 can potentially release (a) angiostatin 4.5 from plasminogen, (b) "cystatin-like domain 3" from low molecular weight kininogen, and (c) fibrinopeptide B and peptide beta15-42 from the Bbeta chain of fibrinogen. hK5 could also play a role in the regulation of the binding of plasminogen activator inhibitor 1 to vitronectin. Our findings suggest that hK5 may be implicated in tumor progression, particularly in invasion and angiogenesis, and may represent a novel therapeutic target.     

Activation and enzymatic characterization of recombinant human kallikrein 8

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM.     

Intracellular proteolytic activity of cathepsin B is associated with capillary-like tube formation by endothelial cells in vitro

The lysosomal cysteine protease cathepsin B is implicated in degradation of extracellular matrix (ECM), a crucial step in a variety of physiological and pathological processes, including tumor dissemination and angiogenesis. In this study, we analyzed the contribution of extracellular and intracellular cathepsin B activity on the formation of capillary-like tubular structures by human umbilical vein endothelial cells (HUVECs) grown on Matrigel matrix, using general and specific cysteine protease inhibitors. We demonstrated, by confocal assay using quenched fluorescent protein substrate DQ-collagen IV, that endothelial cells degrade ECM both intracellularly and pericellularly. Intracellular cathepsin B activity detected by degradation of Z-Arg-Arg cresyl violet substrate was co-localized with the products of DQ-collagen IV degradation in the perinuclear region and in the capillary-like tubular structures. Treatment of cells with membrane-permeable CA-074 Me effectively abolished intracellular cathepsin B activity, and resulted in reduced tube length (32.3+/-9.4% at 10 microM), total tubule area (49.6+/-12.4% at 10 microM), and the number of branch points of tubules (47.5+/-7.7% at 10 microM) in a dose-dependent manner. In contrast, CA-074 (0.1-10 microM), a membrane-impermeable cathepsin B specific inhibitor, general cysteine protease inhibitors chicken cystatin (5 microM) and E-64 (10 microM), and the metalloprotease inhibitor Minocycline (10 microM) showed no significant inhibitory effect in our angiogenesis model. These results show that, besides multiple regulatory molecules, intracellular cathepsin B also contributes to the neovascularization process and should be considered as a potential therapeutic target.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Expression and localization of SerpinB11 in mouse uteri during peri-implantation and the estrous cycle

Serine protease inhibitor (Serpin) B11 has been identified as a novel serine protease inhibitor but the biological functions of SerpinB11 in female reproduction are unknown. Therefore, we investigate the spatiotemporal expression and regulation of SerpinB11 during the peri-implantation period. SerpinB11 mRNA and protein were detected in the uteri of pregnant mice on days 1–8 (day 1?=?presence of a vaginal plug). SerpinB11 protein was localized in the embryonic implantation site on day 5 when embryo implantation occurred and was also strongly expressed in the primary decidual zone on day 6 and secondary decidual zone on days 7 and 8. The expression of SerpinB11 was induced by the activated blastocyst (based on patterns of expression during pseudopregnancy and delayed implantation) and by artificially induced decidualization. Moreover, expression of SerpinB11 was regulated by estradiol and progesterone in ovariectomized mice. The results were further supported by data from the estrous cycle. Thus, SerpinB11 is probably involved in embryo implantation and decidualization.     

Baculoviral expression and characterization of rodent cathepsin S

The cysteinyl proteinase cathepsin S is implicated as a key enzyme in the processing of major histocompatability complex (MHC) class II molecules expressed on antigen presenting cells and thus is a potential therapeutic target for modulation in immune system-based disease. We have identified a form of rat cathepsin S, similar to a published mouse form with an eight-amino acid extended presequence relative to the human enzyme and the previously published rat enzyme. In addition, we have expressed these mouse and rat proteins in baculovirally infected Sf9 insect cells along with "humanized" forms truncated by eight residues at the amino-terminus. All forms of the rodent proteinases were overexpressed and milligram per litre amounts of functional enzyme could be isolated from the cells and/or the cell culture supernatant. Furthermore, addition of a carboxy-terminal hexahistidine purification tag had no effect on the kinetic characteristics of any of the enzyme forms against the Boc-Val-Leu-Lys-AMC peptide substrate (rat k(cat) s(-1) approximately 30; mouse k(cat) s(-1) approximately 65). Differences were seen in the potency of the generic cysteine proteinase inhibitor, E64, against the human, mouse, or rat form of the enzyme (13.3 x 10(4), 43.2 x 10(4), and 25 x 10(4) K(obe)/[I] M(-1) s(-1), respectively). Such data highlights the need for greater awareness of species variation in inhibitor potency. These reagents are vital for confirming inhibitor potency against the endogenous form of the enzyme prior to evaluation of drug candidates in rodent model systems.     

Serine proteinase inhibitor 3 and murinoglobulin I are potent inhibitors of neuropsin in adult mouse brain

Extracellular serine protease neuropsin (NP) is expressed in the forebrain limbic area of adult brain and is implicated in synaptic plasticity. We screened for endogenous NP inhibitors with recombinant NP (r-NP) from extracts of the hippocampus and the cerebral cortex in adult mouse brain. Two SDS-stable complexes were detected, and after their purification, peptide sequences were determined by amino acid sequencing and mass spectrometry, revealing that target molecules were serine proteinase inhibitor-3 (SPI3) and murinoglobulin I (MUG I). The addition of the recombinant SPI3 to r-NP resulted in an SDS-stable complex, and the complex formation followed bimolecular kinetics with an association rate constant of 3.4 +/- 0.22 x 10(6) M(-1) s(-1), showing that SPI3 was a slow, tight binding inhibitor of NP. In situ hybridization histochemistry showed that SPI3 mRNA was expressed in pyramidal neurons in the hippocampal CA1-CA3 subfields, as was NP mRNA. Alternatively, the addition of purified plasma MUG I to r-NP resulted in an SDS-stable complex, and MUG I inhibited degradation of fibronectin by r-NP to 24% at a r-NP/MUG I molar ratio of 1:2. Immunofluorescence histochemistry showed that MUG I localized in the hippocampal neurons. These findings indicate that SPI3 and MUG I serve to inactivate NP and control the level of NP in adult brain, respectively.     

Amplification effect and mechanism of action of ET-1 in U-46619-induced vasoconstriction in pig skin

The aim of this study was to investigate if a low concentration of endothelin-1 (ET-1; 8 x 10(-10) M) may amplify the skin vasoconstrictor effect of other vasoactive substances in the pathogenesis of skin vasospasm. Pig skin flaps (6 x 16 cm) were perfused with Krebs buffer equilibrated with 95% O(2) and 5% CO(2) at 37 degrees C and pH 7.4. Skin perfusion pressure measured by a pressure transducer and skin perfusion assessed by the dermofluorometry technique were used for assessment of skin vasoconstriction. We observed that ET-1 (8 x 10(-10) M) significantly amplified the concentration-dependent (10(-7)-10(-5) M) skin vasoconstrictor effect of norepinephrine. More importantly, we observed for the first time that this low concentration of ET-1 also amplified the concentration-dependent (10(-8)-10(-6) M) skin vasoconstrictor effect of the thromboxane A(2) mimetic U-46619, and this amplification effect of ET-1 was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine (5 x 10(-6) M). Conversely, the PKC activator phorbol 12,13-dibutyrate (10(-7) M) amplified the vasoconstrictor effect of U-46619. Furthermore, the sensitivity of the skin vasculature to the vasoconstrictor effect of extracellular Ca(2+) in U-46619-induced skin vasoconstriction was significantly enhanced in the presence of 8 x 10(-10) M ET-1. Finally, the cyclooxygenase inhibitor indomethacin (5 x 10(-6) M) did not affect the amplification effect of ET-1 on U-46619-induced skin vasoconstriction. We conclude that a low concentration of ET-1 can amplify the skin vasoconstrictor effect of U-46619 independent of endogenous cyclooxygenase products, and the mechanism may involve activation of PKC and increase in sensitivity of the contractile apparatus to Ca(2+) in smooth muscle cells.     

Structural investigation of the alpha-1-antichymotrypsin: prostate-specific antigen complex by comparative model building.

Prostate-specific antigen (PSA), produced by prostate cells, provides an excellent serum marker for prostate cancer. It belongs to the human kallikrein family of enzymes, a second prostate-derived member of which is human glandular kallikrein-1 (hK2). Active PSA and hK2 are both 237-residue kallikrein-like proteases, based on sequence homology. An hK2 model structure based on the serine protease fold is presented and compared to PSA and six other serine proteases in order to analyze in depth the role of the surface-accessible loops surrounding the active site. The results show that PSA and hK2 share extensive structural similarity and that most amino acid replacements are centered on the loops surrounding the active site. Furthermore, the electrostatic potential surfaces are very similar for PSA and hK2. PSA interacts with at least two serine protease inhibitors (serpins): alpha-1-antichymotrypsin (ACT) and protein C inhibitor (PCI). Three-dimensional model structures of the uncleaved ACT molecule were developed based upon the recent X-ray structure of uncleaved antithrombin. The serpin was docked both to PSA and hK2. Amino acid replacements and electrostatic complementarities indicate that the overall orientation of the proteins in these complexes is reasonable. In order to investigate PSA's heparin interaction sites, electrostatic computations were carried out on PSA, hK2, protein C, ACT, and PCI. Two heparin binding sites are suggested on the PSA surface and could explain the enhanced complex formation between PSA and PCI, while inhibiting the formation of the ACT-PSA complex, PSA, hK2, and their preliminary complexes with ACT should facilitate the understanding and prediction of structural and functional properties for these important proteins also with respect to prostate diseases.     

Participation of bone-marrow stem cells in the differentiation of mdx mice striated muscle

Two sets of experiments were carried out. The first one involved chimeric mice, obtained by intravenously injections of bone marrow derived cells taken from transgenic C57BL/6 mice, expressing GFP, to 5 Gy X-ray irradiated mdx or C57BL/6 mice. In 2 months M. quadriceps femoris of chimeric mice were destroyed by surgical clamp. Following the next 4-5 weeks, the same muscles were studied for the presence of GFP-positive striated muscle fibres. In the case of chimeric C57BL/6 mice GFP-positive striated muscle fibres were observed in 0.3 +/- 0.5 and in 0.2 +/- 0.3 % of destroyed muscle, and in lateral (control) muscle, consequently. In the case of chimeric mdx mice, positive results were observed in 1.7 +/- 0.4 and in 0.5 +/- 0.3 % of destroyed and control muscles, respectively. In the second set of experiments, the GFP-positive bone marrow cells were used for multiple intramuscular injections to M. quadriceps femoris of C57BL/6 or mdx mice in a dose of 2 x 10(5)-5 x 10(5) cells per mouse. Before injection, GFP-positive bone marrow cells were fractionated in a 63 % Percoll solution and then were exhausted from differentiated cells by magnetic manner using CD4, CD8, CD38, CD45R, CD119, Ly-6G, and F4/80 antibodies. After 2-3 weeks, as many as 0.15 +/- 0.40 and 0.1 +/- 0.2 % of GFP-positive muscle fibres were found in injected and control muscles of C57BL/6 mice, respectively. In the case of mdx mice, the frequency of GFP-positive striated muscle fibres was 2.0 +/- 0.8 and 1.2 +/- 0.6 % for injected and control muscles, respectively. A conclusion is made that bone marrow stem cells can take part in differentiation of mdx mouse muscles after their delivery by needle injections.     

Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2-15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B(1) and B(2) receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B(1) and B(2) receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.