A potential role for protease nexin 1 overexpression in the pathogenesis of scleroderma. (1/188)

Scleroderma currently affects approximately 75,000-100,000 individuals in the United States. Fibroblasts isolated from lesional skin of scleroderma patients overexpress collagens and other matrix components, and this abnormality is maintained for multiple passages in culture. To understand the molecular basis for matrix gene overexpression, we performed a differential display comparison of fibroblasts from clinically lesional and nonlesional scleroderma skin. The results suggested that protease nexin 1 (PN1), a protease inhibitor, is overexpressed in scleroderma fibroblasts. Northern blot verification showed that lesional and nonlesional scleroderma fibroblasts had three- to five-fold increased levels of PN1 mRNA compared with healthy fibroblasts. Western analysis showed that scleroderma fibroblasts also secreted more PN1. In situ hybridization of skin biopsy specimens demonstrated PN1 expression in the dermis of four out of six scleroderma patients but no PN1 expression in the dermis of six healthy volunteers. Transient or stable overexpression of PN1 in mouse 3T3 fibroblasts increased collagen promoter activity or endogenous collagen transcript levels, respectively. PN1 mutagenized at its active site and antisense PN1 both failed to increase collagen promoter activity. These results suggest that overexpression of enzymatically active PN1 may play a pathogenic role in the development of the scleroderma phenotype.  (+info)

Transforming growth factor-beta1 as a regulator of the serpins/t-PA axis in cerebral ischemia. (2/188)

The tissue type plasminogen activator (t-PA) is a serine protease that is involved in neuronal plasticity and cell death induced by excitotoxins and ischemia in the brain. t-PA activity in the central nervous system is regulated through the activation of serine protease inhibitors (serpins) such as the plasminogen activator inhibitor (PAI-1), the protease nexin-1 (PN-1), and neuroserpin (NSP). Recently we demonstrated in vitro that PAI-1 produced by astrocytes mediates the neuroprotective effect of the transforming growth factor-beta1 (TGF-beta1) in NMDA-induced neuronal cell death. To investigate whether serpins may be involved in neuronal cell death after cerebral ischemia, we determined, by using semiquantitative RT-PCR and in situ hybridization, that focal cerebral ischemia in mice induced a dramatic overexpression of PAI-1 without any effect on PN-1, NSP, or t-PA. Then we showed that although the expression of PAI-1 is restricted to astrocytes, PN-1, NSP, and t-PA are expressed in both neurons and astrocytes. Moreover, by using semiquantitative RT-PCR and Western blotting, we observed that only the expression of PAI-1 was modulated by TGF-beta1 treatment via a TGF-beta-inducible element contained in the PAI-1 promoter (CAGA box). Finally, we compared the specificity of TGF-beta1 action with other members of the TGF-beta family by using luciferase reporter genes. These data show that TGF-beta and activin were able to induce the overexpression of PAI-1 in astrocytes, but that bone morphogenetic proteins, glial cell line-derived neutrophic factor, and neurturin did not. These results provide new insights into the regulation of the serpins/t-PA axis and the mechanism by which TGF-beta may be neuroprotective.  (+info)

The mRNA for protease nexin-1 is expressed in human dermal papilla cells and its level is affected by androgen. (3/188)

Protease nexin-1, an inhibitor of serine proteases, plays important parts in the regulation of the growth, differentiation, and death of cells by modulating proteolytic activity. The mRNA for protease nexin-1 accumulates in rat dermal papilla cells in a hair cycle-dependent fashion and its levels are well correlated with the ability of dermal papilla cells to support hair growth. In an attempt to characterize the potential role of protease nexin-1 as a modulator of hair growth in humans, we investigated the steady-state level of protease nexin-1 mRNA in cultured human dermal papilla cells using a semiquantitative technique that involved reverse transcription and polymerase chain reaction, as well as the localization of this mRNA in vivo using dissected hair follicles. Protease nexin-1 mRNA was expressed in all dermal papilla cells examined, and it was also identified in the lower part of the connective tissue sheath. Moreover, we found that levels of protease nexin-1 mRNA were depressed by dihydrotestosterone, the most potent androgen, in cultured dermal papilla cells obtained from balding scalp. Our results suggest that protease nexin-1 might be a key molecule in the control of hair growth in humans and, moreover, that the androgen-mediated downregulation of the synthesis of protease nexin-1 might be associated with the progression of male-pattern baldness.  (+info)

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1. (4/188)

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.  (+info)

Serpins in the human hair follicle. (5/188)

Proteinases and their inhibitors are very likely to function as mediators or regulators of the hair growth cycle. Very little information is currently available, however, regarding the specific inhibitors present in human hair follicles at defined stages of their growth cycle. In this study we have analyzed two proteinase inhibitors, plasminogen activator inhibitor type 2 and protease nexin 1, in human hair follicles using in situ hybridization and/or immunohistochemistry. Protease nexin 1 mRNA was found only in the mesenchymal population of the hair follicle, i.e., the follicular papilla cells, during the anagen but not the catagen phase. In contrast, plasminogen activator inhibitor type 2 was localized to several epithelial populations in the follicle: the more differentiated cells of the infundibulum; the companion layer in anagen follicles; and the single layer of outer root sheath cells directly abutting the club hair in telogen follicles. At least some of the plasminogen activator inhibitor type 2 in human follicles appears to be in the relaxed form, as evidenced by strong staining with an antibody that is specific for this form of the inhibitor. This suggests that plasminogen activator inhibitor type 2 interacts with and is cleaved by an endogenous follicular proteinase and supports a constitutive role for this inhibitor in human follicular epithelia.  (+info)

Roles of the heparin and low density lipid receptor-related protein-binding sites of protease nexin 1 (PN1) in urokinase-PN1 complex catabolism. The PN1 heparin-binding site mediates complex retention and degradation but not cell surface binding or internalization. (6/188)

We have previously described thrombin (Th)-protease nexin 1 (PN1) inhibitory complex binding to cell surface heparins and subsequent low density lipid receptor-related protein (LRP)-mediated internalization. Our present studies examine the catabolism of urinary plasminogen activator (uPA)-PN1 inhibitory complexes, which, unlike Th.PN1 complexes, bind almost exclusively through the uPA receptor. In addition, the binding site in PN1 required for the LRP-mediated internalization of Th.PN1 complexes is not required for the LRP-mediated internalization of uPA.PN1 complexes. Thus, the protease moiety of the complex partially determines the mechanistic route of entry. Because cell surface heparins are only minimally involved in the binding and internalization of uPA.PN1 complexes, we then predicted that complexes between uPA and the heparin binding-deficient PN1 variant, PN1(K7E), should be catabolized at the same rate as complexes formed with native PN1. Surprisingly, the uPA.PN1(K7E) complexes were degraded at only a fraction of the rate of native complexes. Internalization studies revealed that both uPA. PN1(K7E) and native uPA.PN1 complexes were initially internalized at the same rate, but uPA.PN1(K7E) complexes were rapidly retro-endocytosed in an intact form. By examining the pH dependence of complex binding in the range of 4.0-7.0, it was determined that the uPA.PN1 inhibitory complexes must specifically bind to endosomal heparins at pH 5.5 to be retained and sorted to lysosomes. These studies are the first to document a role for heparins in the catabolism of SERPIN-protease complexes at a point further in the pathway than cell surface binding, and this role may extend to other heparin-binding LRP-internalized ligands.  (+info)

Sexually dimorphic expression of protease nexin-1 and vanin-1 in the developing mouse gonad prior to overt differentiation suggests a role in mammalian sexual development. (7/188)

The mammalian sex-determining pathway is controlled by the presence or absence of SRY expression in the embryonic gonad. Expression of SRY in males is believed to initiate a pathway of gene expression resulting in testis development. In the absence of SRY, ovary development ensues. Several genes have now been placed in this pathway but our understanding of it is far from complete and several functional classes of protein appear to be absent. Sex-determining genes frequently exhibit sexually dimorphic patterns of expression in the developing gonad both before and after overt differentiation of the testis or ovary. In order to identify additional sex-determining or gonadal differentiation genes we have examined gene expression in the developing gonads of the mouse using cDNA microarrays constructed from a normalized urogenital ridge library. We screened for genes exhibiting sexually dimorphic patterns of expression in the gonad at 12.5 and 13.5 days post-coitum, after overt gonad differentiation, by comparing complex cDNA probes derived from male and female gonadal tissue at these stages on micro-arrays. Using in situ hybridization analysis we show here that two genes identified by this screen, protease nexin-1 (Pn-1) and vanin-1 (Vnn1), exhibit male-specific expression prior to overt gonadal differentiation and are detected in the somatic portion of the developing gonad, suggesting a possible direct link to the testis-determining pathway for both genes.  (+info)

SERPIN regulation of factor XIa. The novel observation that protease nexin 1 in the presence of heparin is a more potent inhibitor of factor XIa than C1 inhibitor. (8/188)

In the present studies we have made the novel observation that protease nexin 1 (PN1), a member of the serine protease inhibitor (SERPIN) superfamily, is a potent inhibitor of the blood coagulation Factor XIa (FXIa). The inhibitory complexes formed between PN1 and FXIa are stable when subjected to reducing agents, SDS, and boiling, a characteristic of the acyl linkage formed between SERPINs and their cognate proteases. Using a sensitive fluorescence-quenched peptide substrate, the K(assoc) of PN1 for FXIa was determined to be 7.9 x 10(4) m(-)(1) s(-)(1) in the absence of heparin. In the presence of heparin, this rate was accelerated to 1.7 x 10(6), M(-)(1) s(-)(1), making PN1 a far better inhibitor of FXIa than C1 inhibitor, which is the only other SERPIN known to significantly inhibit FXIa. FXIa-PN1 complexes are shown to be internalized and degraded by human fibroblasts, most likely via the low density lipoprotein receptor-related protein (LRP), since degradation was strongly inhibited by the LRP agonist, receptor-associated protein. Since FXIa proteolytically modifies the amyloid precursor protein, this observation may suggest an accessory role for PN1 in the pathobiogenesis of Alzheimer's disease.  (+info)

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