Báo cáo khoa học: The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury

The Calcium-Binding Domain of the Stress Protein SEP53 Is Required for Survival in Response to Deoxycholic Acid-Mediated Injury

Tác giả: Joanne Darragh, Mairi Hunter, Elizabeth Pohler, Lenny Nelson, John F. Dillon, Rudolf Nenutil, Borek Vojtesek, Peter E. Ross, Neil Kernohan, and Ted R. Hupp

Lĩnh vực: Sinh học phân tử, Nghiên cứu ung thư.

Nội dung tài liệu: Nghiên cứu này tập trung vào vai trò của protein sốc SEP53, một protein 53 kDa có chức năng chưa xác định được tìm thấy trong biểu mô vảy của thực quản. Nghiên cứu đã phát triển các phương pháp đo lường tác động của axit deoxycholic (DCA) lên các phản ứng do SEP53 điều hòa đối với tổn thương. Kết quả cho thấy SEP53 có thể hoạt động như một yếu tố sinh tồn trong các dòng tế bào động vật có vú, có khả năng làm giảm quá trình chết theo chương trình tế bào do DCA gây ra và làm giảm sự gia tăng canxi nội bào do DCA gây ra. Việc loại bỏ miền liên kết canxi EF-hand bảo tồn cao của SEP53 làm mất hoạt tính sinh tồn của protein và khả năng bảo vệ khỏi tổn thương do DCA, đồng thời cho phép tăng canxi khi có thách thức từ DCA. Những phát hiện này chỉ ra rằng SEP53 có thể điều chỉnh sự xâm nhập canxi do DCA gây ra và xác định một con đường sinh tồn mới có thể làm sáng tỏ các cơ chế liên quan đến tổn thương tế bào vảy và sự phát triển ung thư liên quan.

Mục lục chi tiết:

• The calcium-binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid-mediated injury
• Keywords
• Correspondence
• Present address
• Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.
• Human cancers develop through a multistage process involving morphological changes in tissue, mutations in oncogenes and tumour suppressor genes, and epigenetic programmes that give rise to enhanced survival in a stressed microenvironment [1]. The development of human cancer is proving to be a tissue-specific process involving an interaction between mutated cells and the unique conditions within a particular local matrix and microenvironment. Such local cellular stresses include hypoxia, acidification, pro-oxidants from the diet, genome instability and altered autocrine responses. This evolutionary path relies on the developing tumour cell to repair, survive and overcome intrinsic tumour-suppressing signals that normally are used to kill abnormal cells and maintain tissue integrity. The mechanisms underlying tissue-specific responses to local environment in cancer development are largely undefined.
• Abbreviations
• Results
• SEP53 protein is expressed in human squamous epithelium
• Having previously shown, using a functional proteomics approach, that SEP53 is one of the major proteins induced by ex vivo stress to normal squamous epithelium [17], we needed to confirm that the SEP53 protein is in fact expressed in normal human squamous epithelium of the oesophagus. We first needed to develop antibodies to SEP53 and the human SEP53 gene was cloned into a bacterial and insect cell-expression system for the purification and acquisition of full-length protein for immunization, and to develop a panel of monoclonal antibodies (MAb). A tryptic digest of pure full-length SEP53 protein (Fig. 1A, lane 1) gave rise to a ladder of bands (as in Fig. 1A, lane 2) that was used to define the number of unique MAb clones. Three distinct classes of MAbs were grouped according to binding activity to different tryptic fragments (Fig. 1A, lanes 2, 4, 6, 8, and 10). Class A MAb produced a unique pattern of immunoreactive bands (Fig. 1A, lane 2) that was distinct from Class B MAb (Fig. 1A, lane 4), whilst the Class C MAb epitope was destroyed by the trypsinization as effectively no ladder of bands was produced (Fig. 1A, lane 6, 8 and 10).
• We next investigated whether SEP53 protein was expressed in squamous epithelium using these immunochemical reagents. The SEP53 protein is highly expressed in normal squamous epithelium under conditions in which Anterior Gradient-2 is relatively low (Fig. 1B, Normal). As a control for the integrity of the Barrett’s cell population, the Anterior Gradient-2 protein is confirmed to be highly overexpressed in Barrett’s samples [21] compared with normal squamous epithelium from the same patient (Fig. 1D, Barrett’s versus Normal). SEP53 immunostaining can also be observed in the suprabasal layer of squamous epithelium (Fig. 1F), where immunoreactivity is generally cytoplasmic granular staining with minor epimembranous staining in maturing and mature squamous cells. Furthermore, SEP53 is variably expressed in Barrett’s
• where Anterior Gradient-2 protein is relatively high (Fig. 1B, Barrett’s). However, this expression of SEP53 enriched in biopsies endoscopically defined as Barrett’s epithelium might be due to a contamination of normal squamous epithelium in the biopsy. The variable expression of the acid- and glucose-regulated SEP70 protein [17] (Fig. 1C, Barrett’s), under conditions where SEP53 protein is variable (Fig. 1B, Barrett’s), highlights heterogeneity in the Barrett’s samples with respect to all three stress proteins. Nevertheless, the SEP53 protein is in fact expressed in normal human squamous epithelium and this prompted us to continue studying the gene to define a possible molecular function for the protein in stress-responsive pathways.
• Developing cell models to examine effects of DCA on cell death
• SEP53 was originally identified as a protein synthesized ex vivo after heat or ethanol stress [17]. The physiological stress the SEP53 responds to in cells is, however, undefined, as heat exposure to the oesophagus and ethanol are unlikely to be evolutionary adaptations. The oesophagus is an organ that is commonly exposed to bile acids and the structure of normal oesophageal epithelium is altered by bile exposure [22]. Developing knowledge of the effects that these chemicals may have on oesophageal epithelial cells and apoptotic pathways might be relevant to understanding the molecular function of SEP53. We were therefore interested in determining whether the SEP53 gene had any effects on modifying DCA-induced cell stresses. However, prior to examining the effects of DCA on SEP53-mediated apoptotic responses, we wanted to confirm that DCA was in fact a significant constituent of gastric fluid.
• To analyse gastric fluid samples for bile acid content, bile acids were extracted, derivatized and then analysed by gas chromatography. The relative retention times of peaks present in the gastric fluid sample
• were calculated (Fig. 2A) and the relevant bile acid peaks identified by comparison with values from the standard mix of pure lipids (data not shown). Bile acids were detected in 92% (158/172) of patient samples and the concentration and/or composition of the bile acid pool varied considerably between patient samples (Fig. 2B). In samples with detectable levels, the concentration of total bile acids ranged from 1 µm to 6.4 mm, with a mean of 323 µm (Fig. 2B). In total, 31% of samples contained no or low concentrations of bile acids, with 32% having high concentrations in excess of 200 µm, and the remaining 37% of cases having concentrations ranging between 20 and 200 µm (Fig. 2C,D). The majority of patient samples contained a mixture of bile acids (as well as cholesterol, Fig. 2E), including DCA, chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA) and cholic acid (CA), with both conjugated and unconjugated (Fig. 2B,E) forms being identified. The primary bile acids, CA and CDCA, with mean concentrations of 118 and 112 µm, respectively, were present in a higher concentrations than the secondary bile acids, with the mean concentration of DCA being 63 µm and LA levels averaging 17 µm (Fig. 2B). The proportion of DCA to CA in gastric juice was higher than anticipated (Fig. 2E), as in normal duodenal fluid the DCA levels have been found to be one fifth of cholate [23].
• DCA was present in gastric samples and the range of DCA was from 1 µm to over 1.5 mm (Fig. 2B). The physiological levels of DCA that are associated with injury are not known, as patients fast before entering the clinic for sample collection. Furthermore, it is not known whether chronic exposure to low levels that are not acutely toxic induces a worse or better indicator than single supratoxic acute doses over time. Despite this heterogeneity in bile levels in gastric fluid, it is difficult to extrapolate to in vivo concentrations, however,
• using rabbit oesophageal mucosa as a model, the epithelium concentrates bile acids up to 7x lumenal concentrations [24]. Thus, given the range of DCA in patients (1 μm to > 1 mm) and given that bile can be concentrated from the lumen up to 7x [24], the possible concentration of DCA in cells might be from 7 μm to 10 mm. Furthermore, Zhang et al. [25] evaluated the range of bile acids (as in Fig. 2) and found that ~500 µm of selected bile acids were required to give rise to significant apoptosis. These latter levels were in the range we used (Figs 2 and 3) and given this, we titrated DCA from low µm to > 1 mm to determine
• whether it was toxic in our cell assays and whether it was modified by SEP53.
• We next evaluated the effects of these key bile acids present in gastric fluid on the cell-cycle parameters a set of relatively well-characterized oesophageal cancer cell lines (OE21, KYSE 30, OE 19 and OE33), particularly to determine whether DCA was able to significantly induce injury. In the presence of DCA up to a concentration of 500 µm, no significant apoptotic response was obtained in the OE21 or KYSE 30 squamous cell lines [Fig. 3A and C versus Fig. 4G (OE21 cells)], in contrast to the oesophageal cancer Eca109
• cell line in which this dose gives rise to 22% apoptotic cells [25]. The adenocarcinoma cell lines (OE 19 and OE33 cells) did, however, demonstrate a dose-dependent death response following exposure to DCA (Fig. 3B,D versus control Fig. 4A,G). The production of these sub-G1 fragments detected by FACS after DCA exposure was confirmed to be apoptotic by characteristic nuclear morphology changes (Fig. 4M,N and Q,R). Titration of DCA up to 500 µm demonstrated a dose-dependent increase in sub-G1 fragments which can be observed selectively in OE33 and OE19 cells (Fig. 3E) and is consistent with data published recently in a different oeopshageal cancer cell line [25].
• DCA-mediated apoptosis is mediated by a PKC-dependent pathway and is p53 independent
• One of the principal biological functions of the tumour-suppressor protein p53 is as a mediator of apoptosis in response to cellular stress and DNA damage [26]. Because DCA can induce DNA damage [14], the role of p53 in mediating DCA-dependent apoptosis was investigated using a pair of isogenic p53+ and p53 cell lines [27], in order to determine whether we needed to consider the p53 status in dissecting DCA-mediated signalling. The HCT116 (p53+) isogenic cell line was incubated with increasing concentrations of DCA (0-500 μμ) for 6 h and the resultant stressed cells were then fixed, stained with PI and the mean (± SEM) (n = 3) percentage of apoptotic cells measured by flow cytometry (Fig. 3F-H). Under these conditions, apoptosis was elevated, in a dose-dependent manner, from 2 to 58% of the cell population as defined by sub-G1 fragments. Both of the HCT116 (p53+ and p53¯) cell lines were equally sensitive to DCA-induced apoptosis (data not shown) indicating that DCA-induced apoptosis does not require signalling via p53 in these colonic cell lines. Furthermore, because the OE33 and OE19 cell lines have mutant p53 (data not shown), p53-independent apoptosis operates under these conditions.
• In order to define a positive mechanism for DCA-mediated apoptosis in OE33 versus OE21 cells, we evaluated a set of common protein kinase inhibitors for an attenuation of the response in OE33 cells (data not shown). One striking observation was made using the protein kinase C (PKC) inhibitor bisindolylmaleimide I (Bis-I), which inhibited DCA-dependent apoptosis (Fig. 31). The control inactive version of the inhibitor bisindolylmaleimide V (Bis-V) was unable to block the apoptosis (Fig. 3J), demonstrating the selectivity in the response. Because the PKC pathway was being activated to induce apoptosis in the OE33 cell line, but not in the OE21 cell line, we reasoned that differential activation of key components of the PKC pathway, the pro-apoptotic GSK3 or pro-survival PKB kinases might account for the altered DCA-mediated apoptotic response [28,29]. Consistent with this,
• DCA attenuated phosphorylation of the normally pro-survival PKB at the activating site of PKB in OE33 cells (Fig. 3K, lane 4 versus 2). By contrast, basal inactivating phosphorylation of GSK3 was reduced in OE33 cells (Fig. 3M, lane 4 versus 2). The opposite occurs in the OE21 cells: DCA did not block phosphorylation of PKB in the resistant OE21 cells (Fig. 30, lane 4 versus 2), although GSK phosphorylat-ion actually increased in OE21 cells (Fig. 3Q, lane 4 versus 2). The data suggest that the GSK3-PKB-PKC
• pathway axis, rather than p53, is a primary mediator of the differential apoptotic response of the two cell lines.
• Gastric fluid contains a mixture of different bile acids in addition to DCA (as in Fig. 2B). These have different biochemical properties and in terms of biological effect they have been shown to vary in their ability to induce apoptosis in colorectal cancer cell lines, although DCA is the prime bile used in generalized research [11,13,25,30]. Therefore, the effect of several conjugated and unconjugated bile acids on the induction of apoptosis in both the sensitive OE33 and resistant OE21 oesophageal cell lines was investigated (Fig. 4). The sensitivity of the adenocarcinoma cell line, OE33 to deoxycholic acid-induced apoptosis was abrogated when this bile acid was conjugated to taurine (taurodeoxycholic acid; Fig. 4B versus Fig. 4D). Similarly the addition of CA, a trihydroxy bile acid or ursodeoxycholic (UDCA) a 3a:7ẞ dihydroxy bile acid had no damaging effect on OE33 cells (Fig. 4C,D). However, CDCA and LA did induce apoptosis in the OE33 cell line, with the percentage of sub-G1 cells increasing to 25 and 23%, respectively (Fig. 4E,F). Furthermore, the levels of apoptosis induced by these two bile acids were similar to levels obtained following a DCA stress in this same cell line (25%, Fig. 3D,E versus Fig. 4A). OE21 cells remained resistant to all bile acids studied, irrespective of their hydrophobicity (Fig. 4G-L). Thus, CDCA, DCA and LA were the three most potent cell death-inducers and the mean concentration of these in gastric fluid was 112, 63 and 17 μμ, respectively. The data indicate that DCA is in fact the second-most abundant toxic effector, exerts a similar toxicity to the other two bile acids, and affirms its use as a model damaging agent.
• SEP53 functions as a survival factor in a clonogenic assay
• The key stresses thought to predominate in oesophageal squamous epithelium and cause tissue injury include heat shock [31], low pH [5] and DCA [14]. We examined specifically whether SEP53 protein modifies the DCA death response, as this is proving to be a physiologically relevant DNA damaging agent [14,25]. We had first analysed a range of tumour cell lines for SEP53 protein levels and have not found one cell that expressed the protein including the OE panel described here (data not shown). This may relate to the fact that the SEP53 gene is located on chromosome 1q21 within a group of proteins named the epidermal differentiation complex fused-gene family and that this locus might be silenced by chromatin remodelling as part of
• a general mechanism that suppresses genes from this locus in cancer cells [19,2