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Clusterin proteins: stress-inducible polypeptides with proposed functions in multiple organ dysfunction

Departments of ¹ Radiation Oncology, ² Pharmacology and ³ Biochemistry, Laboratory of Molecular Stress Responses, Case School of Medicine and the Case Comprehensive Cancer Center, Case Western Reserve University, 2103 Cornell Road WRB 3-531 North, Cleveland, OH 44106-7285, USA

View larger version (19K): [in a new window]   Figure 1. Synthesis of secretory and nuclear clusterin proteins. (a) Synthesis of secretory clusterin (sCLU) protein forms is depicted. Full length clusterin (CLU) mRNA is translated with a leader polypeptide that targets the protein to endoplasmic reticulum (ER). The ER polypeptide is cleaved and a 60 kDa pre-secretory clusterin (psCLU) protein is produced. As the protein is transported to the Golgi complex, it is cleaved into and polypeptides, which are heavily glycosylated and form a heterodimeric complex via five disulfide bonds. Ultimately, an 80 kDa protein is excreted from the cell. (b) Intracellular CLU protein is produced from a truncated, alternatively spliced nuclear clusterin (nCLU) mRNA, which lacks the ER-targeting domain [14]. Translation of the nCLU mRNA results in 49 kDa pre-nuclear clusterin (pnCLU) protein. This 49 kDa pnCLU is dormant in the cytoplasm, presumably by hiding the nuclear localisation site (NLS) within the C-terminal portion of the protein through homodimerisation or self-folding of the protein; pnCLU contains two coiled-coil domains that interact and cause dormancy. After significant stress (>1 Gy), pnCLU is "activated" and altered post-translationally, forming an 55 kDa cell death protein. Expression of the C-terminus of this nCLU protein was sufficient to cause cell death [14].

View larger version (16K): [in a new window]   Figure 2. Clusterin (CLU) gene expression is induced by changes in endoplasmic reticulum stress, caused by thapsigargin (TG) or high dose BAPTA-AM, agents that cause extensive changes in Ca2+ homeostasis. (a) Changes in survival (measured by colony-forming ability; see Materials and Methods section) in mock-treated cells compared with cells that were pre-treated with 3 µM BAPTA-AM. (b) Changes in endogenous 60 kDa pre-secretory clusterin (psCLU) as well as 40 kDa secretory clusterin (sCLU) levels were monitored in untreated (no treatment, NT) or TG-exposed (2–20 nM) MCF-7 cells. Whole cell protein extracts were prepared at 24 h or 72 h and probed for CLU protein responses by standard Western blot procedure as described previously [11, 13]. Induction of endogenous protein levels was observed by as little as 2 nM TG, which did not cause a significant lethality response (see (a)). Cells harvested 24 h and 72 h after 5 Gy ionising radiation (IR) were used as a positive control. All experiments were performed three or more times, and representative Western blots are shown.

View larger version (29K): [in a new window]   Figure 3. Thapsigargin (TG) induces clusterin (CLU) promoter activity. Log-phase 1403 MCF-7 cells, which contain a stably integrated CLU promoter–firefly luciferase reporter construct [15], were exposed to various doses of TG (0–12 nM) and monitored for changes in CLU promoter activity at 24 h, 48 h, 72 h and 96 h post treatment. CLU promoter activities were monitored by standard luminometer assays, as described previously [15, 16]. Cells harvested at 24 h, 48 h, 72 h and 96 h after 10 Gy ionising radiation were used as a positive control. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. NT, no treatment.

View larger version (33K): [in a new window]   Figure 4. Altered Ca2+ homeostasis is sufficient to induce clusterin (CLU) gene expression. Two experiments were performed using 1403 MCF-7 cells: (1) increasing doses of BAPTA-AM (0–20 µM) were administered with a single dose of thapsigargin (TG) (10 nM); and (2) cells were pre-treated with one dose of BAPTA-AM (3 µM) followed by subsequent increasing doses of TG (0–20 nM, 1 h). CLU promoter activity was assessed as described in Figure 3 and in the Materials and Methods section. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. NT, no treatment.

View larger version (14K): [in a new window]   Figure 5. BAPTA-AM exposures at high doses cause increased clusterin (CLU) promoter activities. 1403 MCF-7 cells were pre-loaded with vehicle alone (0.1% dimethylsulfoxide (DMSO)) or with BAPTA-AM in vehicle for 30 min, and then mock irradiated or exposed to 5 Gy ionising radiation (IR). CLU promoter activity was then assessed by standard luminometer readings as described in the Materials and Methods section. Experiments were performed three times in triplicate, and statistical analyses of the data were performed using the Student's t-test. At high doses, BAPTA-AM can cause Ca2+ influx and induce CLU promoter activity to a level statistically equivalent to 5 Gy IR. High dose BAPTA-AM exposures also resulted in increases in endogenous CLU protein responses (not shown).

View larger version (27K): [in a new window]   Figure 6. Two-hit theory of initiation of multiple organ dysfunction (MOD). The initial trauma to a tissue or organ is considered the initial event. Reactive oxygen species (ROS), lipid peroxidation, released Ca2+ from the endoplasmic reticulum (ER) and liberated cytokines are proposed to mediate cell death reactions in initially affected cells as well as in neighbouring cells and tissue that may or may not have been initially affected by the traumatic stress. The "second hit" is thought to be initiated by activation of inflammatory cells and genesis of cell debris or secondary infections and endotoxaemia. Uncontrolled systemic inflammatory responses (SIRS) and immunological dissonance then initiates MOD in localised tissues and organs, and globally throughout the body. See text for further discussion.

View larger version (24K): [in a new window]   Figure 7. Clusterin (CLU) gene products are proposed to be major players in defence and initiation responses to trauma that directly influence multiple organ dysfunction (MOD). CLU gene activation is a molecular switch between life and death. At low doses, secretory clusterin (sCLU) production provides a cytoprotective molecular chaperone defence mechanism in clearing cell debris from traumatised tissue. At higher doses, the pre-nuclear form of CLU (pnCLU, a form of the protein that resides dormant in the cytoplasm of most cells) becomes activated through an as yet uncharacterised post-translational mechanism. Activated nuclear clusterin (nCLU), a pro-death mature 55 kDa protein that causes apoptosis, is formed. Widespread activation of nCLU in organs after MOD-causing traumatic insults then facilitates multiple organ failure. See text for further discussion. ROS, reactive oxygen species.