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Kazumi Ishidoh

Publications and source records attributed to Kazumi Ishidoh.

17 recordsLinked to original sources
PubMed2006

Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest.

In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20-day bedrest to clarify ubiquitin-dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross-sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real-time reverse transcription-polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl-b and atrogin-1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl-b- or atrogin-1-mediated ubiquitination plays an important role in unloading-induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

UNLABELLED: Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. INTRODUCTION: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. MATERIALS AND METHODS: Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. RESULTS: In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. CONCLUSIONS: These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.

Animals↗
PubMed2005

Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress.

We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.

Animals↗
PubMed2005

Gene expression profiles in response to Fas stimulation in Trypanosoma cruzi-infected host cells.

To determine the molecular mechanism by which apoptosis is inhibited in Trypanosoma cruzi-infected host cells, we used human cDNA apoptosis chips to compare the gene expression profiles in response with 'death ligands target' (Fas) stimulation in infected and uninfected cells. Of the 164 apoptosis-related genes examined, 20, including those encoding both pro- and anti-apoptotic proteins, were highly up-regulated in the infected group. Genes encoding caspases and apoptosis inhibitors were optimally expressed 10-30 min after induction of apoptosis, whereas genes involved in transcriptional regulation and cell proliferation were up-regulated after 2-24 h. These results suggest that host anti-apoptotic gene(s) may play a crucial role in the inhibition of Fas-mediated apoptosis in T. cruzi-infected cells.

Animals↗
PubMed2004

Podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin L and alpha3 integrin.

Podocyte foot process effacement and disruption of the slit diaphragm are typically associated with glomerular proteinuria and can be induced in rats by the injection of puromycin aminonucleoside. Here, we show that the induction of puromycin aminonucleoside nephrosis involves podocyte migration conducted by a coordinated interplay between the cysteine protease cathepsin L and alpha(3) integrin. Puromycin aminonucleoside treatment up-regulates cathepsin L expression in podocytes in vivo as well as expression and enzymatic activity of cathepsin L in podocytes in vitro. Isolated podocytes from mice lacking cathepsin L are protected from cell puromycin aminonucleoside-induced cell detachment. The functional significance of cathepsin L expression was underscored by the observation that puromycin aminonucleoside-induced cell migration was slowed down in cathepsin L-deficient podocytes and by the preservation of cell-cell contacts and expression of vital slit diaphragm protein CD2AP. Cathepsin L expression and activity were induced in podocytes lacking alpha(3) integrin. Similarly, acute functional inhibition of alpha(3) integrin in wild type podocytes with a blocking antibody increased the expression of cathepsin L activity. Down-regulation of alpha(3) integrin protected against puromycin aminonucleoside-induced podocyte detachment. In summary, these data establish that podocyte foot process effacement is a migratory event involving a novel interplay between cathepsin L and alpha(3) integrin.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B.

SKD1 belongs to the AAA-ATPase family and is one of the mammalian class E Vps (vacuolar protein sorting) proteins. Previously we have reported that the overexpression of an ATPase activity-deficient form of SKD1 (suppressor of potassium transport growth defect), SKD1(E235Q), leads the perturbation of membrane transport through endosomes and lysosomes, however, the molecular mechanism behind the action of SKD1 is poorly understood. We have identified two SKD1-binding proteins, SBP1 and mVps2, by yeast two-hybrid screening and we assign them as mammalian class E Vps proteins. The primary sequence of SBP1 indicates 22.5% identity with that of Vta1p from Saccharomyces cerevisiae, which was recently identified as a novel class E Vps protein binding to Vps4p. In fact, SBP1 binds directly to SKD1 through its C-terminal region (198-309). Endogenous SBP1 is exclusively localized to cytosol, however it is redirected to an aberrant endosomal structure, the E235Q compartment, in the cells expressing SKD1(E235Q). The ATPase activity of SKD1 regulates both the membrane association of, and assembly of, a large hetero-oligomer protein complex, containing SBP1, which is potentially involved in membrane transport through endosomes and lysosomes. The N-terminal half (1-157) of human SBP1 is identical to lyst-interacting protein 5 and intriguingly, SKD1 ATPase activity significantly influences the membrane association of lyst protein. The SKD1-SBP1 complex, together with lyst protein, may function in endosomal membrane transport. A primary sequence of mVps2, a mouse homologue of human CHMP2A/BC-2, indicates 44.4% identity with Vps2p/Did4p/Chm2p from Saccharomyces cerevisiae. mVps2 also interacts with SKD1 and is localized to the E235Q compartment. Intriguingly, the N-terminal coiled-coil region of mVps2 is required for the formation of the E235Q compartment but not for binding to SKD1. We propose that both SBP1 and mVps2 regulate SKD1 function in mammalian cells.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2004

Skeletal muscle gene expression in space-flown rats.

Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients.

PURPOSE: To identify differentially expressed genes in human keratoconus keratocyte by cDNA microarray. METHODS: Normal and keratoconic cornea were cultured for keratocytes. RNA was extracted. cDNA probe labeled with fluorescence dye was made from Poly A+ RNA, hybridized with microarray slide, containing 164 human apoptosis genes. Signal intensity was measured. Expression ratio between keratoconus and normal was determined using ImaGene Ver.3.0. Identified genes were further evaluated by RT-PCR and real-time PCR. RESULTS: Five over-expressed and four under-expressed genes were identified. Of these, differential expression of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), human insulin-like growth factor binding protein 5 (IGFBP5), and IGFBP3 were verified and confirmed by RT-PCR. Real-time PCR showed TNFAIP6 increased by 3.3 folds, while IGFBP5, IGFBP3 decreased by 14 and 11 folds respectively. CONCLUSIONS: The identified genes could be important and deserve further investigation. Significant differential expression of TNFAIP6, IGFBP5, and IGFBP3 may indicate an important role of these genes in the mechanism underlying stromal thinning.

Adult↗
PubMed2004

Inflammatory response and cathepsins in silica-exposed Hermansky-Pudlak syndrome model pale ear mice.

Hermansky-Pudlak syndrome (HPS) is a hereditary disorder involving the sorting processes of intracellular organelles such as lysosomes of reticuloendothelial cells. Pale ear (ep) mouse is known to have the HPS1 gene mutation, which is seen in patients with HPS and pulmonary fibrosis. As pulmonary fibrosis is not spontaneously observed in ep mice, we hypothesized that external stimuli are necessary for the genetic predisposition of its development. We used silica as the external stimulus to induce the alveolar macrophage-mediated inflammatory response and evaluated the pathological changes of the lung and biochemical analysis of collagenolytic lysosomal enzymes cathepsins L and B in ep mice. Treatment with silica induced the following: persistent accumulation of activated macrophages; delayed clearance of silica from alveolar spaces; and increased collagen fibers in alveolar tissues, which were shown with trichrome staining in ep mice. The comparison of bronchoalveolar lavage cells between the naïve ep and control mice revealed: decreased enzymatic activities but increased antigenic levels of cathepsins L and B, resulting in significantly lower ratios of activity to antigen; increased ceroid deposits and cathepsin L antigens in lysosomes; and no abnormal forms of cathepsins were detected. After silica instillation, activities of cathepsin L in the ep mice increased but ratios of activity to antigen were still significantly low. These phenomena induced by silica suggest that external stimuli bring forth fibrogenesis in the animal models or humans that have HPS1 gene mutation.

Animals↗
PubMed2004

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages.

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.

Animals↗
PubMed2003

The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2003

Fibroblast growth factor-inducible 14 mediates multiple pathways of TWEAK-induced cell death.

TWEAK, a TNF family member, is produced by IFN-gamma-stimulated monocytes and induces multiple pathways of cell death, including caspase-dependent apoptosis, cathepsin B-dependent necrosis, and endogenous TNF-alpha-mediated cell death, in a cell type-specific manner. However, the TWEAK receptor(s) that mediates these multiple death pathways remains to be identified. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which was responsible for TWEAK-induced proliferation of endothelial cells and angiogenesis. Because Fn14 lacks the cytoplasmic death domain, it remains unclear whether Fn14 can also mediate the TWEAK-induced cell death. In this study, we demonstrated that TWEAK could induce apoptotic cell death in Fn14 transfectants. A pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, rather sensitized the Fn14 transfectants to TWEAK-induced cell death by necrosis via reactive oxygen intermediates and cathepsin B-dependent pathway. By using newly generated agonistic anti-Fn14 mAbs, we also observed that Fn14 is constitutively expressed on the cell surface of all TWEAK-sensitive tumor cell lines, and can transmit the multiple death signals. Moreover, an anti-Fn14 mAb that blocks TWEAK-Fn14 interaction could totally abrogate TWEAK binding and TWEAK-induced cell death in all TWEAK-sensitive tumor cell lines. These results revealed that the multiple pathways of TWEAK-induced cell death are solely mediated by Fn14.

Animals↗
PubMed2002

Multiple pathways of TWEAK-induced cell death.

TWEAK, a recently identified member of the TNF family, is expressed on IFN-gamma-stimulated monocytes and induces cell death in certain tumor cell lines. In this study, we characterized the TWEAK-induced cell death in several tumor cell lines that exhibited distinct features. Although the TWEAK-induced cell death in Kym-1 cells was indirectly mediated by TNF-alpha and was inhibited by cycloheximide, the TWEAK-induced cell death in HSC3 cells or IFN-gamma-treated HT-29 cells was not inhibited by anti-TNF-alpha mAb or cycloheximide, suggesting a direct triggering of cell death via TWEAK receptor in the latter cell lines. The TWEAK-induced apoptosis in HSC3 cells and IFN-gamma-treated HT-29 cells was associated with caspase-8 and caspase-3 activation. Although a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, inhibited the TWEAK-induced cell death in HSC3 cells, it rather sensitized HT-29 cells to TWEAK-induced cell death by necrosis. This necrosis was abrogated by lysosomal proteinase inhibitors, particularly a cathepsin B inhibitor, [L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester. During the process of TWEAK-induced necrosis, cathepsin B was released from lysosome to cytosol. Although DR3 has been reported to be a receptor for TWEAK, all TWEAK-sensitive tumor cell lines used in this study did not express DR3 at either protein or mRNA level, but did bind CD8-TWEAK specifically. These results indicated that TWEAK could induce multiple pathways of cell death, including both caspase-dependent apoptosis and cathepsin B-dependent necrosis, in a cell type-specific manner via TWEAK receptor(s) distinct from DR3.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Sulfhydryl oxidase (SOx) from mouse epidermis: molecular cloning, nucleotide sequence, and expression of recombinant protein in the cultured cells.

Skin sulfhydryl oxidase (SOx) is an enzyme that catalyzes disulfide (S-S) cross-linking through the oxidation of sulfhydryl compounds in the skin. In this study, using the enzyme purified from rat seminal vesicle, we obtained peptide sequences for SOx by mass spectrometry. We then searched for SOx nucleotides corresponding highly to the rat peptide sequences by assembling murine-expressed sequence tags (ESTs) from the GeneBank database. The assembled mouse SOx cDNA has an open reading frame of 1704-bp nucleotides, translating into a size of 568 amino acids. The calculated molecular mass of the mouse SOx protein is 65 kDa. This mouse sequence can be amplified from total RNAs of various mouse tissue samples by reverse transcription polymerase chain reaction, especially highly amplified from those of the seminal vesicles and epidermis. The cDNA fragment was subsequently cloned into the mammalian expression vector (pTARGET-MSSOx), allowing us to express mouse recombinant SOx protein in cultured cells. When pTARGET-MSSOx was transfected, Western blot analysis using anti-SOx antiserum could detect a 65 kDa-band of recombinant SOx in both samples from the whole cell extract and the medium after the harvest of the HEK cells. In immunohistochemical analysis, the Pt-K2 cells, following the introduction of pTARGET-MSSOx, seemed to generate a SOx protein reactive to anti-SOx antiserum in the cells. Moreover, the indirect staining of the S-S bonds using N-(7-dimethylamino-4-methyl coumarinyl) maleimide (DACM), following the addition of N-ethylmaleimide and dithiothreitol, showed that the formation of S-S bridges almost matched the localization of SOx expression in the Pt-K2 cells after the transfection. In essence, we cloned skin SOx cDNA and characterized it as one of the S-S cross-linking enzymes. The SOx clone from mouse epidermis seems to be useful for investigating the potential function of the enzyme in the epidermis, especially for understanding the physiological role of SOx in the differentiation of keratinocytes.

Amino Acid Sequence↗
PubMed2002

Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes.

UNLABELLED: Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes. BACKGROUND: Podocyte damage is considered to be an important factor in the development of glomerulosclerosis. Morphological studies on experimental models of progressive glomerular disease have identified the detachment of podocytes from the glomerular basement membrane (GBM) as a critical step in the development and progression of glomerulosclerosis. Degradation of the GBM by proteinases also might be a potential mechanism of the detachment because the process impairs the connection between podocytes and the GBM. The present study examined the effects of basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor (PDGF) on the secretion of proteinases [cathepsin L and matrix metalloproteinases (MMPs)] and their inhibitors [cystatin C and tissue inhibitor of metalloproteinase-2 (TIMP-2)] from differentiated podocytes in culture. METHODS: Expression of mRNAs for receptors of growth factors (bFGF, PDGF, TGF-beta1), the proteinases and their inhibitors in differentiated podocytes were shown by RT-PCR. The secretion of cathepsin L, cystatin C and TIMP-2 from differentiated podocytes were shown by immunoblot analysis. The activities of MMPs-2 and -9 from differentiated podocytes were shown by gelatin zymography. RESULTS: Expression of mRNAs for receptors of the growth factors, the proteinases and their inhibitors were confirmed. bFGF increased the secretion of cathepsin L (5.04-fold at 20 ng/mL), but did not alter the secretion of its extracellular inhibitor, cystatin C. In contrast, TGF-beta1 increased the activities of MMPs-2 and -9 (3.23-fold at 10 ng/mL and 25.3-fold at 10 ng/mL, respectively) from differentiated podocytes, but did not enhance the secretion of its inhibitor, TIMP-2. In addition, bFGF enhanced the secretion of TIMP-2 (2.75-fold at 20 ng/mL) and TGF-beta1 enhanced the secretion of cystatin C (2.32-fold at 20 ng/mL). These results demonstrate the imbalance of the secretion of proteinases and their inhibitors after incubation of such growth factors. Of particular interest was the observation of differences in regulation of proteinases and their extracellular inhibitors in response to bFGF and TGF-beta1. PDGF only slightly increased the secretion of cathepsin L (2.54-fold at 20 ng/mL) but exerted no effect on the secretion of cystatin C, MMPs, and TIMP-2 from differentiated podocytes. CONCLUSION: These results indicate, to our knowledge for the first time, that in differentiated podocytes, both cathepsin L and its inhibitor are independently regulated by different growth factors. It appears that increases in proteolytic activities may induce degradation of the glomerular basement membrane (GBM), which plays an important role in the progression of glomerulosclerosis.

Animals↗
PubMed2002

Processing and activation of lysosomal proteinases.

Lysosomal proteinases are translated as preproenzymes, transferred through the Golgi apparatus as proenzymes, and localized in lysosomes as the mature enzymes. Pulse-chase analyses and the immunoisolation of proenzymes or recombinant proenzymes are useful tools for analyzing this process, but the processing proteinases that participate in this pathway are largely unknown. Recently, we developed a new method for analyzing processing proteinases using Bafilomycin A1 and proteinase inhibitors. Here we summarize the recent progress including our results obtained using this method.

Animals↗