Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation

https://doi.org/10.1016/S0303-7207(98)00051-3Get rights and content

Abstract

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bc1-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.

Introduction

The induction of cell death in lymphocytes exposed to glucocorticosteroid hormones has intrigued investigators for over 50 years (Dougherty and White, 1943, Heilman and Kendall, 1944). Understanding the mechanism is of considerable pragmatic importance as glucocorticoids are commonly used to treat a broad range of lymphoid malignancies. Glucocorticoid-induced cell death is initiated by interaction of the glucocorticoid hormone with its cognate receptor, a transcription factor which functions to either induce or repress gene transcription (Baxter et al., 1971). The genes specifically involved in mediating the cell death decision have not been identified and there is conflicting evidence regarding the relative importance of transcriptional repression versus transcriptional induction in this process (Dieken and Miesfeld, 1992, Helmberg et al., 1995). Nevertheless, once the cells have been committed to die, they undergo an apoptotic form of cell death characterized by condensation of nuclear chromatin and DNA fragmentation (Wyllie, 1980).

The process of apoptosis is highly conserved from the nematode, C. elegans, to man and involves a systematic, stepwise process of cellular destruction regulated by genes encoding either death inhibitors or effectors (Steller, 1995). The C. elegans gene ced-9 encodes an inhibitor of cell death, whereas the ced-3 and ced-4 genes encode death effectors. The mammalian homologue of ced-9, bc1-2, is a member of a family of genes encoding proteins that function either to inhibit or promote cell death (Cory, 1995). Bc1-2 itself inhibits cell death induction by diverse signals, indicating that it acts in a common pathway where multiple death signals converge. The mammalian homologue of ced-3 is the cysteine protease, interleukin 1-β converting enzyme (ICE) (Yuan et al., 1993). Ced-3 is required for execution of apoptotic cell death in C. elegans, suggesting the possibility that ICE or related proteases might similarly be required for apoptosis in mammalian cells (Yuan et al., 1993). ICE, along with up to ten ICE-like proteases, compose a family of cysteine proteases, recently referred to as caspases (Alnemri et al., 1996), that characteristically cleave proteins at aspartic acid residues (Fraser and Evan, 1996, Henkart, 1996). These proteases fall into several subfamilies, including the Ced-3 family, which includes the mammalian protease CPP32 (caspase-3, Yama or apopain) and the ICE (caspase-1) family. Recent findings suggest that caspases operate within an amplifiable protease cascade, culminating in activation of caspase-3 and related caspases (Martin and Green, 1995, Fraser and Evan, 1996). Cleavage of selected target proteins by caspase-3 and related proteases appears to be directly responsible for the stereotypic morphological changes characteristic of apoptosis (Martin and Green, 1995), while caspase-mediated cleavage of poly(ADP-ribose) polymerase (PARP) serves as a marker of caspase activation during apoptosis (Kaufmann et al., 1993, Lazebnik et al., 1994, Nicholson et al., 1995).

Based on studies in ICE-deficient mice, ICE itself is not required for glucocorticoid-induced apoptosis of thymocytes (Kuida et al., 1995), but PARP is cleaved during the process of glucocorticoid-induced apoptosis of thymocytes, indicating that caspases are activated by glucocorticoid treatment (Kaufmann et al., 1993). In addition, other classes of proteases have been implicated as mediators of cell death in glucocorticoid-treated lymphocytes. For example, recent studies have implicated the calcium-dependent neutral protease calpain as a mediator of cell death in glucocorticoid-treated lymphocytes (Squier et al., 1994), while other studies based primarily on the use of protease inhibitors have implicated both cysteine proteases and serine proteases as mediators of apopotsis in thymocytes (Weaver et al., 1993, Fearnhead et al., 1995, Hara et al., 1996).

Therefore, the role of caspases in mediating apoptosis of WEHI7.2 mouse lymphoma cells induced by the glucocorticoid hormone dexamethasone (DX) and the endoplasmic reticulum (ER) calcium-ATPase inhibitor thapsigargin (TG) was investigated. DX and TG represent widely divergent cell death initiating signals that appear to converge into a common Bc1-2 regulated pathway (Lam et al., 1994, Distelhorst and McCormick, 1996). In the present studies, PARP cleavage was used as an index of in vivo caspase activity following DX and TG treatment in both wild type WEHI7.2 cells, which do not express Bc1-2, and in the stable WEHI7.2 transfectant, W.Hb12, which expresses a high level of Bc1-2 (Lam et al., 1994). The role of caspases in apoptosis induction was further characterized in vivo by Western blotting and in cell extracts by assessing the cleavage of in vitro synthesized p35, a baculovirus protein cleaved by many caspases, including caspase-3 (Bump et al., 1995). The requirement for caspases in DX and TG induced cell death in vivo was tested using the cell permeant caspase inhibitors, Z-VAD-fmk and Z-DEVD-fmk, compared to inhibitors of other classes of proteases implicated in apoptosis, concluding cathepsins and calpains. Based on the findings, it was concluded that caspase-3 is activated in a Bcl-2-regulated fashion by both DX and TG, although with markedly different kinetics. Furthermore, caspase activation is required for apoptosis induction by both DX and TG.

Section snippets

Materials

DX and other chemicals were from Sigma. TG was purchased from LC Laboratories. Culture medium was from BioWhittaker. Horse serum was obtained from Hyclone. l-Glutamine, antibiotics and non-essential amino acids were from Gibco/BRL. N-Acetyl-leucyl-leucyl-norleucinal (Calpain inhibitor I) and N-acetyl-leucyl-leucyl-methional (Calpain inhibitor II) were purchased from Boehringer-Mannheim. Benzyloxycarbonyl-Val-Ala-Asp(o-methyl)-fluoromethylketone (Z-Val-Ala-Asp(o-Me)-CH2F; Z-VAD-fmk),

Results

In experiments reported here the WEHI7.2 and W.Hbl2 lines were employed. WEHI7.2 is a mouse T cell lymphoma line that lacks Bc1-2 (Lam et al., 1994). WEHI7.2 was stably transfected with an expression vector, pSFFV-Bc1-2, deriving a series of clones expressing high, low and intermediate levels of Bc1-2 (Lam et al., 1994). There is a direct relationship between the level of Bc1-2 expressed in various subclones and resistance to DX- and TG-induced apoptosis (Lam et al., 1994and unpublished data).

Discussion

The findings reported here show that caspase activation is involved in mediating apoptosis in mouse lymphoma cells following treatment with DX or TG. Indeed, the data indicate that caspase activity is required for induction of apoptotic cell death. Recently, Robertson et al (Robertson et al., 1997) described the inhibition of glucocorticoid-induced apoptosis and caspase-3 activation in a human T cell leukemia line by overexpression of baculovirus p35. Thus, different experimental approaches in

Acknowledgements

The authors thank Paul Friesen and Robert Horvitz for providing expression vectors and Kristy Kikly for providing antibody to caspase-3. This work was supported by National Institutes of Health grants RO1 CA42755-08 and T32 CA59366.

References (46)

  • S.J. Martin et al.

    Protease activation during apoptosis: Death by a thousand cuts?

    Cell

    (1995)
  • C.E. Milligan et al.

    Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro

    Neuron

    (1995)
  • M. Muzio et al.

    FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex

    Cell

    (1996)
  • J. Yuan et al.

    The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme

    Cell

    (1993)
  • H. Zhu et al.

    An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells

    FEBS Lett.

    (1995)
  • J.D. Baxter et al.

    Glucocorticoid receptors in lymphoma cells in culture: relationship to glucocorticoid killing activity

    Science

    (1971)
  • N.J. Bump et al.

    Inhibition of ICE family proteases by baculovirus antiapoptotic protein p35

    Science

    (1995)
  • S. Cory

    Regulation of lymphocyte survival by the Bc1-2 gene family

    Ann. Rev. Immunol.

    (1995)
  • E.S. Dieken et al.

    Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis

    Mol. Cell. Biol.

    (1992)
  • T.F. Dougherty et al.

    Effect of pituitary adrenotropic hormone on lymphoid tissue

    Proc. Soc. Exp. Biol. Med.

    (1943)
  • D.R. Dowd et al.

    Stable expression of the calbindin-D28K complementary DNA interferes with the apoptotic pathway in lymphocytes

    Mol. Endocrinol.

    (1992)
  • Duke, R.C., Cohen, J.J., 1992. Morphological and biochemical assays of apoptosis. In: Coligan, J.E., Kruisbeck, A.M.,...
  • R.A. Flomerfelt et al.

    Recessive mutations in a common pathway block thymocyte apoptosis induced by multiple signals

    J. Cell Biol.

    (1994)
  • Cited by (59)

    • Oxidative damage to osteoblasts can be alleviated by early autophagy through the endoplasmic reticulum stress pathway - Implications for the treatment of osteoporosis

      2014, Free Radical Biology and Medicine
      Citation Excerpt :

      Next, Mc3T3-E1 cells were pretreated with 5 mM 3-MA, 10 nM Baf-A1, 100 nM Rap, or vehicle for 1 h, and then exposed to 1 mM H2O2 for 6 h. Flow cytometric analysis of the cells demonstrated that the apoptosis incidence significantly increased when autophagy was inhibited by 3-MA or Baf-A1 treatment, while a significant decrease of the apoptosis incidence was observed when autophagy was stimulated by Rap treatment (Fig. 3C). Apoptosis was characterized with activation of caspase-3 and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP) [50]. Therefore, cleaved PARP and cleaved caspase-3 (cleaved Cas-3) were assessed to further evaluate the effect of the H2O2-induced autophagy on apoptosis in the osteoblasts.

    View all citing articles on Scopus
    View full text