Biomedical subjects

W Declercq

Publications and source records attributed to W Declercq.

At least 37 records · Page 2Linked to original sources

PubMed2000

Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.

Programmed cell death of epidermal keratinocytes (KC) results in the formation of cornified cells, which constitute the outermost skin layer, the stratum corneum. Here we show by reverse transcription-polymerase chain reaction, western blot, and immunohistochemistry that epidermal KC express caspase-14, a member of the caspase family of pro-apoptotic proteases, in a tissue-specific manner. Caspase-14 protein abundance strongly increases during terminal differentiation of KC in vivo and in vitro. Under conditions that lead to stratum corneum formation caspase-14 cleavage products, which indicate proenzyme activation, appeared in the KC lysates. Cleavage of the enzyme was also detected in lysates from normal human epidermis and in extracts of stratum corneum. Our findings demonstrate that caspase-14 is activated during KC differentiation and strongly suggest that it is involved in the formation of the human skin barrier.J Invest Dermatol 115:1148-1151 2000

Caspase 14↗

PubMed1999

More than one way to die: apoptosis, necrosis and reactive oxygen damage.

Cell death is an essential phenomenon in normal development and homeostasis, but also plays a crucial role in various pathologies. Our understanding of the molecular mechanisms involved has increased exponentially, although it is still far from complete. The morphological features of a cell dying either by apoptosis or by necrosis are remarkably conserved for quite different cell types derived from lower or higher organisms. At the molecular level, several gene products play a similar, crucial role in a major cell death pathway in a worm and in man. However, one should not oversimplify. It is now evident that there are multiple pathways leading to cell death, and some cells may have the required components for one pathway, but not for another, or contain endogenous inhibitors which preclude a particular pathway. Furthermore, different pathways can co-exist in the same cell and are switched on by specific stimuli. Apoptotic cell death, reported to be non-inflammatory, and necrotic cell death, which may be inflammatory, are two extremes, while the real situation is usually more complex. We here review the distinguishing features of the various cell death pathways: caspases (cysteine proteases cleaving after particular aspartate residues), mitochondria and/or reactive oxygen species are often, but not always, key components. As these various caspase-dependent and caspase-independent cell death pathways are becoming better characterized, we may learn to differentiate them, fill in the many gaps in our understanding, and perhaps exploit the knowledge acquired for clinical benefit.

Animals↗

PubMed1999

Proteolytic cleavage of beta-catenin by caspases: an in vitro analysis.

Cleavage of structural proteins by caspases has been associated with the severe morphological changes occurring during the apoptotic process. One of the proteins regulating the connection of the actin filament with cadherins in a cell-cell adhesion complex is beta-catenin. During apoptosis, both an N-terminal and a small C-terminal part are removed from beta-catenin. Removal of the N-terminal part may result in a disconnection of the actin filament from a cadherin cell-cell adhesion complex. We demonstrate that caspase-8, -3 and -6 directly proteolyse beta-catenin in vitro. However, the beta-catenin cleavage products generated by caspase-8 were different from those generated by caspase-3 or caspase-6. Caspase-1, -2, -4/11 and -7 did not or only very inefficiently cleave beta-catenin. These data suggest that activation of procaspase-3, -6 or -8 by different stimuli in the cell might result in a differential proteolysis of beta-catenin.

Animals↗

PubMed1999

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin.

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.

Anthracenes↗

PubMed1999

Identification of caspases that cleave presenilin-1 and presenilin-2. Five presenilin-1 (PS1) mutations do not alter the sensitivity of PS1 to caspases.

Mutations in the presenilin (PS) genes PSI and PS2 are involved in Alzheimer's disease (AD). Recently, apoptosis-associated cleavage of PS proteins was identified. Here we demonstrate that PS1 as well as PS2 are substrates for different members of the caspase protein family. Remarkably, the caspases acting on PS1 could be subdivided in two groups. One group, containing caspase-8, -6 and -11, cleaved PSI after residues ENDD329 and to a lesser extent after residues AQRD341. A second group consisting of caspase-3, -7 and -1 acted uniquely on AQRD341. Importantly, these two cleavage sites were also recognized by caspases in the C-terminal PS1 fragment produced by constitutive proteolysis. In decreasing order of activity, caspase-8, -3, -1, -6 and -7 proteolysed PS2 at the recognition site D326SYD329. Caspase-8 and -3 exhibited the highest proteolytic activity on both PS1 and PS2. PS1 and PS2 were not hydrolyzed by caspase-2 and PS2 also not by caspase-11. None of five missense mutations affected the sensitivity of PSI to caspase-mediated cleavage. This suggests that AD pathogenesis associated with PS1 missense mutations cannot be explained by a change in caspase-dependent processing.

Animals↗

PubMed1999

Non-specific effects of methyl ketone peptide inhibitors of caspases.

Caspases are a family of cysteine proteases which play a crucial role in apoptosis and inflammation. The involvement of caspases in these processes can be demonstrated by their irreversible inhibition with fluoromethyl ketone and chloromethyl ketone derivatives of peptides resembling the cleavage site of known caspase substrates. These inhibitors irreversibly alkylate the cysteine residue in the active site of caspases. In this study we show that a biotinylated fluoromethyl ketone peptide inhibitor of caspases (z-VAD.fmk) also efficiently affinity-labeled cathepsin B and cathepsin H. In addition, the caspase inhibitors z-VAD.fmk, z-DEVD.fmk and Ac-YVAD.cmk also efficiently inhibited cathepsin B activity in vitro and in tissue culture cells at concentrations that are generally used to demonstrate the involvement of caspases.

Affinity Labels↗

PubMed1999

Immunolocalization of tumor necrosis factor-alpha and its receptors in inflammatory myopathies.

Adhesion molecule upregulation occurs in inflammatory myopathies, and is one of the myriad functions of tumor necrosis factor-alpha (TNF-alpha). TNF-alpha acts via two different receptors of 55 (TNF-R55) and 75 kD (TNF-R75). We immunolocalized TNF-alpha and its receptors in polymyositis, inclusion body myositis and dermatomyositis. In each myopathy, TNF-alpha was detected in macrophages, in myonuclei in regenerating muscle fibers, and freely dispersed in endomysial or perimysial connective tissue. Many endothelial cells in dermatomyositis expressed TNF-alpha. TNF-R55 was strongly expressed on myonuclei of regenerating muscle fibers. TNF-R75 was increased on endothelial cells in the midst of inflammatory infiltrates in each myopathy, and on perifascicular and perimysial endothelia, remote from inflammatory foci in dermatomyositis. Possible TNF-alpha-mediated effects include: increased transendothelial cell trafficking, activation of T/B cells and macrophages, induction of MHC-I gene products, and focal muscle fiber atrophy. In dermatomyositis, the upregulated TNF-R75, via its consensus elements for transcription factors, may be involved in endothelial cell degeneration. Strong TNF-R55 expression on regenerating myonuclei is consistent with a role of TNF-alpha and TNF-R55 in muscle regeneration.

Antigens, CD↗

PubMed1999

The proteolytic procaspase activation network: an in vitro analysis.

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.

Animals↗

PubMed1998

Molecular cloning and identification of murine caspase-8.

Several caspases are mediators of apoptotic cell death. We describe a novel murine member of this growing protein family. Based on homology and especially on the substrate specificity, this new procaspase is identified as the murine counterpart of human procaspase-8. The protein exhibits a rather low similarity (76%) and identity (70%) to human procaspase-8. Procaspase-8 mRNA is expressed in all adult mouse tissues examined, the highest levels being reached in kidney, liver and lung. Procaspase-8 mRNA expression is highest in seven-day old embryos, but also during later stages of development the expression was fairly high. Both human and murine procaspase-8 are very weak substrates for granzyme B as compared to procaspase-3. Murine procaspases-1, 2, 3, 6, 7, 8, 11/4 and 12 are processed by recombinant murine caspase-8, suggesting a key role in the procaspase activation cascade. In addition, murine caspase-8 induced cell death that was inhibited both by cytokine response modifier A and p35. In vitro experiments demonstrated that p35 inhibits caspase-8 directly.

Amino Acid Sequence↗

PubMed1998

Hypericin-induced photosensitization of HeLa cells leads to apoptosis or necrosis. Involvement of cytochrome c and procaspase-3 activation in the mechanism of apoptosis.

Here we report that photoactivated hypericin can induce either apoptosis or necrosis in HeLa cells. Under apoptotic conditions the cleavage of poly(ADP-ribose) polymerase (PARP) into the 85-kDa product is blocked by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (z-DEVD-fmk). Both inhibitors protect cells from apoptosis but cannot prevent hypericin-induced necrosis. Conversely, HeLa cells overexpressing the viral cytokine response modifier A (CrmA), which inhibits caspase-1 and -8, still undergo hypericin-induced apoptosis and necrosis. Evidence is provided for the release of mitochondrial cytochrome c in the cytosol and for procaspase-3 activation in the hypericin-induced cell killing.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Dual signaling of the Fas receptor: initiation of both apoptotic and necrotic cell death pathways.

Murine L929 fibrosarcoma cells were transfected with the human Fas (APO-1/CD95) receptor, and the role of various caspases in Fas-mediated cell death was assessed. Proteolytic activation of procaspase-3 and -7 was shown by Western analysis. Acetyl-Tyr-Val-Ala-Asp-chloromethylketone and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone++ +, tetrapeptide inhibitors of caspase-1- and caspase-3-like proteases, respectively, failed to block Fas-induced apoptosis. Unexpectedly, the broad-spectrum caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone and benzyloxycarbonyl-Asp(OMe)-fluoromethylketone rendered the cells even more sensitive to Fas-mediated cell death, as measured after 18 h incubation. However, when the process was followed microscopically, it became clear that anti-Fas-induced apoptosis of Fas-transfected L929 cells was blocked during the first 3 h, and subsequently the cells died by necrosis. As in tumor necrosis factor (TNF)-induced necrosis, Fas treatment led to accumulation of reactive oxygen radicals, and Fas-mediated necrosis was inhibited by the oxygen radical scavenger butylated hydroxyanisole. However, in contrast to TNF, anti-Fas did not activate the nuclear factor kappaB under these necrotic conditions. These results demonstrate the existence of two different pathways originating from the Fas receptor, one rapidly leading to apoptosis, and, if this apoptotic pathway is blocked by caspase inhibitors, a second directing the cells to necrosis and involving oxygen radical production.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75.

TNF-R55 is the main receptor mediating TNF-induced cytotoxicity. However, in some cells TNF-R75 also signals cell death. In PC60 cells, the presence of both receptor types is required to induce apoptosis following either specific TNF-R55 or TNF-R75 triggering, pointing to a mechanism of receptor cooperation. In this study, we extend previous observations and show that TNF-R55 and TNF-R75 cooperation in the case of apoptosis in PC60 cells is bidirectional. We also demonstrate ligand-independent TNF-R55-mediated cooperation in TNF-R75-induced granulocyte/macrophage-CSF secretion, but not vice versa. To determine which part of the intracellular TNF-R75 sequence was responsible for the observed receptor cooperation in apoptosis, we introduced different TNF-R75 mutant constructs in PC60 cells already expressing TNF-R55. Our data indicate that an intact TNF-R-associated factors 1 and 2 (TRAF1/TRAF2)-binding domain is required for receptor cooperation. These findings suggest a role for the TRAF complex in TNF-R cooperation in the induction of cell death in PC60 cells. Nevertheless, introduction of a dominant negative (DN) TRAF2 molecule was not able to affect receptor cooperation. Remarkably, TRAF2-DN overexpression, which was found to inhibit the TNF-dependent recruitment of endogenous wild-type TRAF2 to the TNF-R75 signaling complex, could neither block TNF-R55- or TNF-R75-induced NF-kappaB activation nor granulocyte/macrophage-CSF secretion. Possibly, additional factors different from TRAF2 are involved in TNF-mediated NF-kappaB activation.

Amino Acid Sequence↗

PubMed1998

Inhibition of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor necrosis factor.

Murine L929 fibrosarcoma cells treated with tumor necrosis factor (TNF) rapidly die in a necrotic way, due to excessive formation of reactive oxygen intermediates. We investigated the role of caspases in the necrotic cell death pathway. When the cytokine response modifier A (CrmA), a serpin-like caspase inhibitor of viral origin, was stably overexpressed in L929 cells, the latter became 1,000-fold more sensitive to TNF-mediated cell death. In addition, TNF sensitization was also observed when the cells were pretreated with Ac-YVAD-cmk or zDEVD-fmk, which inhibits caspase-1- and caspase-3-like proteases, respectively. zVAD-fmk and zD-fmk, two broad-spectrum inhibitors of caspases, also rendered the cells more sensitive, since the half-maximal dose for TNF-mediated necrosis decreased by a factor of 1,000. The presence of zVAD-fmk also resulted in a more rapid increase of TNF-mediated production of oxygen radicals. zVAD-fmk-dependent sensitization of TNF cytotoxicity could be completely inhibited by the oxygen radical scavenger butylated hydroxyanisole. These results indicate an involvement of caspases in protection against TNF-induced formation of oxygen radicals and necrosis.

Amino Acid Chloromethyl Ketones↗

PubMed1998

TRAF2 plays a dual role in NF-kappaB-dependent gene activation by mediating the TNF-induced activation of p38 MAPK and IkappaB kinase pathways.

We previously demonstrated that p38 MAPK is a crucial mediator in the NF-kappaB-dependent gene activation induced by TNF. Here, we have studied the role of several TNF receptor-associated proteins and caspases in p38 MAPK activation by TNF. The latter appears to be dependent on TRAF2, but independent of FADD or caspases. Remarkably, p38 MAPK activation by TNF proceeds independently of the TRAF2-associated NF-kappaB-inducing kinase NIK, which is known to bind and activate two recently identified IkappaB kinases. These results demonstrate that two kinase pathways involved in NF-kappaB regulation, viz. NIK and p38 MAPK-mediated, diverge at the level of TRAF2.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Identification of a new caspase homologue: caspase-14.

Caspases are cysteinyl aspartate-specific proteinases, many of which play a central role in apoptosis. Here, we report the identification of a new murine caspase homologue, viz. caspase-14. It is most related to human/murine caspase-2 and human caspase-9, possesses all the typical amino acid residues of the caspases involved in catalysis, including the QACRG box, and contains no or only a very short prodomain. Murine caspase-14 shows 83% similarity to human caspase-14. Human caspase-14 is assigned to chromosome 19p13.1. Northern blot analysis revealed that mRNA expression of caspase-14 is undetectable in all mouse adult tissues examined except for skin, while it is abundantly expressed in mouse embryos. In contrast to many other caspase family members, murine caspase-14 is not cleaved by granzyme B, caspase-1, caspase-2, caspase-3, caspase-6, caspase-7 or caspase-11, but is weakly processed into p18 and p11 subunits by murine caspase-8. No aspartase activity of murine caspase-14 could be generated by bacterial or yeast expression. Transient overexpression of murine caspase-14 in mammalian cells did not elicit cell death and did not interfere with caspase-8-induced apoptosis. In conclusion, caspase-14 is a member of the caspase family but no proteolytic or biological activities have been identified so far. The high constitutive expression levels in embryos and specific expression in adult skin suggest a role in ontogenesis and skin physiology.

Amino Acid Sequence↗

PubMed1997

Characterization of seven murine caspase family members.

Seven members of the murine caspase (mCASP) family were cloned and functionally characterized by transient overexpression: mCASP-1 (mICE), mCASP-2 (Ich1), mCASP-3 (CPP32), mCASP-6 (Mch2), mCASP-7 (Mch3), mCASP-11 (TX) and mCASP-12. mCASP-11 is presumably the murine homolog of human CASP-4. Although mCASP-12 is related to human CASP-5 (ICErel-III), it is most probably a new CASP-1 family member. On the basis of sequence homology, the caspases can be divided into three subfamilies: first, mCASP-1, mCASP-11 and mCASP-12; second, mCASP-2; third, mCASP-3, mCASP-6 and mCASP-7. The tissue distribution of the CASP-1 subfamily transcripts is more restricted than that of the CASP-3 subfamily transcripts, suggesting that the transcriptional regulation of the CASP members within one subfamily is related, but is quite different between the CASP-1 and the CASP-3 subfamilies. Transient overexpression of each of the seven CASPs induced apoptosis in mammalian cells. Only two, mCASP-1 as well as mCASP-3, were able to process precursor interleukin (IL)-1beta to biologically active IL-1beta. In addition, mCASP-3 is the predominant PARP-cleaving enzyme in vivo.

Amino Acid Sequence↗

PubMed1997

Cleavage of caspase family members by granzyme B: a comparative study in vitro.

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP-1 (interleukin-1 beta-converting enzyme; ICE) subfamily, the CASP-2 (Ich1) subfamily, and the CASP-3 (CPP32) subfamily. Our results reveal that the proforms of the CASP-3 subfamily members mCASP-3 and mCASP-7 are hydrolyzed by granzyme B, while proforms of CASP-2 and CASP-1 subfamily members are not directly cleaved. Only one CASP-3 subfamily member, pro-mCASP-6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP-3 subfamily, but no others, become activated by granzyme B.

Animals↗

PubMed1997

Differential role of calcium in tumour necrosis factor-mediated apoptosis and secretion of granulocyte-macrophage colony-stimulating factor in a T cell hybridoma.

The authors investigated the dependence on extracellular and intracellular free Ca2+ in the induction of apoptosis and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by tumour necrosis factor (TNF) in a rat/mouse T cell hybridoma PC60 R55/R75, using the Ca2+ chelators EGTA and BAPTA/AM, respectively. TNF-induced apoptosis still occurred in the absence of free Ca2+, while GM-CSF production required the continuous presence of Ca2+. The latter was also true for GM-CSF production driven by interleukin 1 (IL-1). The dependence on Ca2+ in the induction of GM-CSF, but not of apoptosis, was further confirmed by the inhibition of TNF- or IL-1-induced cytokine production by cyclosporin A or FK506, drugs that block the Ca2+/calmodulin-dependent protein Ser/Thr phosphatase calcineurin. This differential requirement for Ca2+ illustrates the partial functional redundancy between TNF and IL-1, showing the activation of cytokine gene expression through a Ca(2+)-dependent activation of calcineurin, and a Ca(2+)-independent activation of apoptosis, exerted solely by TNF.

Animals↗