Heat shock protein 70 in apoptosis

The cytoprotective effect provided by heat shock proteins could be shown in many expe-rimental systems (reviewed in: (Beere 2004, 2005; Jäättelä 1999; Samali and Cotter 1996;

Sreedhar and Csermely 2004)). A protective effect in cells exposed to cytotoxic mecha-nisms of the immune system could be demonstrated already quite early. Initially it was shown that a heat shock confers resistance of tumour cells against TNF-α (Gromkowski

2 Introduction

et al. 1989; Jäättelä et al. 1989; Kusher et al. 1990; Sugawara et al. 1990), CTLs (Geginat et al. 1993; Sugawara et al. 1990), and monocytes (Jäättelä and Wissing 1993). Later it was demonstrated that also transfection with heat shock genes including the HSP70 gene can render cells resistant towards TNF-α (Jäättelä et al. 1992, 1998) and other non-immunological stimuli such as UV-irradiation (Simon et al. 1995), ceramide (Ahn et al.

1999), ischemia (Hoehn et al. 2001), or serum withdrawal (Ravagnan et al. 2001).

In the beginning, stress resistance provided by heat shock proteins was mainly attributed to their function as chaperones, which prevent misfolding and aid in re-folding of denatured proteins after stress. Later, it has become obvious that heat shock proteins, including HSP70, interfere with several specific steps of different apoptotic pathways (Dressel and Demiroglu 2006). It was described that HSP70 can prevent the formation of a functional apoptosome by blocking the recruitment of caspase-9 (Beere et al. 2000; Saleh et al. 2000).

Furthermore, HSP70 can suppress the c-Jun-N-terminal kinase (JNK) (stress kinase), which is part of a pro-apoptotic signalling pathway (Bienemann et al. 2008; Gabai et al.

2002, 1997; Mosser et al. 2000). HSP70 can prevent the release of cytotochrome c from mitochondria (Bivik et al. 2007; Mosser et al. 2000) by inhibiting pro-apoptotic molecules such as the BCL-2 family member BAX and thereby stabilises the mitochondrial membrane (Stankiewicz et al. 2005). Moreover, it prevents the nuclear import of AIF (Chaitanya and Babu 2008; Gurbuxani et al. 2003) and inhibits apoptosis by stabilising lysosomes (Dudeja et al. 2009). These are some examples to indicate that HSP70 is able to interfere at several steps of the apoptotic cascade to abrogate cell death. In conclusion, in the stress response system intracellular HSP70 can provide cellular protection.

In accordance with these results HSP70 is found to be overexpressed in many human tumours (Mosser and Morimoto 2004). In some types of tumours, e.g. breast cancer, this correlates with a poor prognosis and resistance against therapy (Garrido et al. 2001;

Jäättelä 1999; Jolly and Morimoto 2000). However, this does not seem to be the case for all kinds of tumours as in osteosarcomas and kidney cell sarcomas the overexpression of HSP70 was found to be associated with a rather good prognosis (Santarosa et al. 1997;

Trieb et al. 1998). This might be due to immunological functions of HSP70.

While intracellular HSP70 in the stress response system mainly protects cells from apop-totic stimuli, extracellular HSP70 can act as an immunological danger signal to activate cells of the innate and adaptive immune system.

HSP70 and other heat shock proteins chaperone antigenic peptides in the cytosol and in the endoplasmic reticulum and prevent their degradation before they are loaded on MHC class I molecules (Srivastava et al. 1998, 1994). Therefore, preparations of HSP70 and other heat shock proteins from virus-infected or tumour cells contain the antigenic repertoire of these cells and can be used as vaccines to stimulate an antigen-specific

im-2 Introduction

mune response (Udono and Srivastava 1993). This is particularly efficient because heat shock protein-peptide complexes are bound to receptors, such as CD91 and lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1), on professional antigen presenting cells (APCs) and are internalised (Basu et al. 2001; Binder et al. 2000; Castellino et al.

2000). The chaperoned peptides are then channeled into the MHC class I presentation pathway and elicit a strong CTL response. In addition, binding of HSP70 to other re-ceptors including Toll-like receptor (TLR)2/4, CD14, CD36, and CD40 can induce the release of pro-inflammatory cytokines and initiate innate immune reactions (Srivastava 2002a, 2002b).

In addition, HSP70 can be translocated to the plasma membrane of stressed cells or is released together with parts of the intact membrane of these cells (exosomes) and stimu-lates macrophages (Vega et al. 2008). When HSP70 is expressed on the cell surface of tumour cells it can function as a recognition structure for NK cells (Gastpar et al. 2004;

Multhoff et al. 1997, 1995). Furthermore, stimulation of NK cells with HSP70 or the HSP70-derived peptide TKD can enhance NK cell-mediated cytotoxicity towards HSP70 plasmamembrane-positive tumours (Multhoff 2002; Multhoff et al. 1997, 1999, 2001).

Stress Response System Immune System

Figure 2.2: Role of HSP70 in the stress response and in the immune systemHSP70 has diverse functions in the stress response and in the immune system. In the stress response system intracellular HSP70 acts as chaperone and protects the cell from cytotoxic stimuli. In tumour cells this protective function of intracellular HSP70 can promote tumourigenicity. On the other hand, in the immune system extracellular HSP70, released from apoptotic or necrotic cells or present on the cell surface of some tumours, acts as “danger signal” and can activate cells of the innate and adaptive immune response.

Thus, HSP70 can incerase immunogenicity. In summary, depending on its location, intracellularly or extracellularly, HSP70 has two opposing functions, which could promote or diminish cancer, respectively.

2 Introduction

In summary as demonstrated in figure 2.2 on the previous page, on the one side HSP70 protects cells by inhibiting apoptosis at many different steps and on the other side it can activate cells of the innate and adaptive immune response when it is released from dying cells or is used as a vaccine.

We were therefore interested in the role of HSP70 in apoptosis induced by cytotoxic cells of the immune system via the granule-exocytosis pathway, which is distinct different from other apoptotic pathways. Would HSP70 be protective against cytotoxic cells although it is able to activate them?

We observed that the rat myeloma cell line 210-RCY3-Ag1.2.3 (Y3) did not express HSP70, even not after heat shock, but that heat shock rendered these cells resistant towards CTLs. The transfer of recombinant HSP70 into Y3 cells abolished the resistance against CTLs (Dressel et al. 2000). This result suggested that HSP70 can improve cell death induced by CTLs in heat-shocked target cells.

The effects of HSP70 were further analysed in the human melanoma cell line Ge. For conditional overexpression of HSP70, the cell line was transfected with a rat Hsp70 gene under the control of a tetracycline-inducible promoter (Ge-tet cells) (Dressel et al. 1999).

For permanent overexpression of HSP70, Ge cells were retrovirally transduced with the same ratHsp70 gene (Ge-Hsp70 cells) (Dressel et al. 2003).

The acute overexpression of HSP70 for 24 hours (hrs) in Ge-tet cells increased the lysis of tumour cells by CTLs using the granule-exocytosis pathway. This effect was neither caused by an increase in MHC class I expression on the cell surface of target cells nor by a function of HSP70 in antigen processing (Dressel et al. 1999). This is in contrast to the findings of Wells et al. (1998) describing that a transfection of the mouse melanoma cell line B16 with HSP70 increased the MHC class I expression and improved thereby recognition and lysis by CTLs. Thus, we assumed that in cytotoxic granule-mediated apoptosis acutely overexpressed HSP70 improves the function of proteins involved in this process. Interestingly, the constitutive overexpression of HSP70 in Ge-Hsp70 cells did not improve susceptibility towards CTLs (Dressel et al. 2003), which implies that the increase in susceptibility does not depend on the level of HSP70 expression but rather on the availibility of additional HSP70. Long-term in contrast to short-term overexpression of HSP70 in Ge-tet cells did also not increase the lysis by CTLs (Dressel et al. 2003). This can be explained by a compensation within the chaperone network down-regulating the constitutively expressed HSC70 upon prolonged overexpression of HSP70 (Dressel et al.

2003). Thus, the increase of susceptibility to CTLs seen in cells acutely overexpressing HSP70 seems to be mediated by HSP70 proteins that are not occupied in physiological functions and are free to chaperone molecules involved in the execution of apoptosis. This concept is supported by the findings that the chaperone HSP60 binds to pro-caspase-3

2 Introduction

and is required for its activation (Samali et al. 1999; Xanthoudakis et al. 1999). Thus, HSP60 acts as a apoptotic protein in this context and HSP70 can also carry-out pro-apoptotic functions under certain circumstances. Furthermore, HSP70 can stabilise the function of CAD, which is able to induce DNA fragmentation in the nucleus, although these findings were reported from TCR-induced T-cells, they show that HSP70 is able to chaperone pro-apoptotic proteins (Liu et al. 2003).

However, so far, nothing is known about the molecular mechanisms that confer the increased sensitivity of Ge-tet cells to CTLs after acute overexpression of HSP70. Inter-estingly, it was described that HSP70 on the cell surface of target cells can directly interact with GrB and also mediate its uptake (Gross et al. 2003b). This suggests that HSP70 might improve specifically the GrB-induced apoptosis.

2.5 Aims

It was the aim of this project to further analyse the role of HSP70 and sulphatase 1 and 2 genes in apoptosis induced by cytotoxic cells of the immune system via the granule-exocytosis pathway.

Previous results had indicated that acute overexpression of HSP70 can augment the lysis of Ge melanoma cells by CTLs using the granule-exocytosis pathway. The molecular mechanism behind this phenomenon is unknown. To further elucidate these pro-apoptotic effects two approaches were followed: (1) To determine whether the cell-death promoting effect of acutely overexpressed HSP70 results from a specific regulation of genes, e.g.

up-regulation of genes encoding for pro or down-regulation of genes encoding for anti-apoptotic proteins, an expression profiling experiment should be performed in the Ge-tet system. (2) To reduce the complexity of killing in the granule-exocytosis pathway, effects of specific effector molecules should be analysed. Priority should be given to GrB because this effector protease is known to interact with HSP70. It should be analysed which key steps in GrB-mediated apoptosis might be affected by HSP70 including the activation of caspases, the loss of the mitochondrial membrane potential ∆Ψ, and DNA fragmentation.

In the second part of the project the role of two other genes in CTL-mediated apoptosis should be investigated. It is known from our work that HSPGs are involved in the uptake of cytotoxic effector molecules such as GrB and control the efficiency of CTL and GrB-induced apoptosis (Raja et al. 2005). Therefore, we planned to investigate the effect of increased sulphation of HSPGs on CTL and GrB-induced apoptosis using cells from mice deficient for theSulf1and Sulf2 genes.

Together the results of this project might contribute to the understanding of factors that control the susceptibility of tumour cells to cytotoxicity mediated by CTLs in the

2 Introduction

granule-exocytosis pathway.

3 Materials