RELT family members activate p38 and induce apoptosis by a mechanism distinct from TNFR1

Abstract

Receptor Expressed in Lymphoid Tissues (RELT) is a human Tumor Necrosis Factor Receptor (TNFR) family member that has two identified homologous binding partners, RELL1 and RELL2. This study sought to further understand the pattern of RELT expression, the functional role of RELT family members, and the mechanism of RELT-induced apoptosis. RELT protein expression was detected in the spleen, lymph node, brain, breast and peripheral blood leukocytes (PBLs). A smaller than expected size of RELT was observed in PBLs, suggesting a proteolytically cleaved form of RELT. RELL1 and RELL2 overexpression activated the p38 MAPK pathway more substantially than RELT in HEK-293 cells, and this activation of p38 by RELT family members was blocked by dominant-negative mutant forms of OSR1 or TRAF2, implicating these molecules in RELT family member signaling. RELT was previously shown to induce apoptosis in human epithelial cells despite lacking the characteristic death domain (DD) found in other TNFRs. Seven deletion mutants of RELT that lacked differing portions of the intracellular domain were created to assess whether RELT possesses a novel DD. None of the deletion mutants induced apoptosis as efficiently as full-length RELT, a result that is consistent with a novel DD being located at the carboxyl-terminus. Interestingly, induction of apoptotic morphology by RELT overexpression was not prevented when signaling by FADD or Caspase-8 was blocked, indicating RELT induces apoptosis by a pathway distinct from other death-inducing TNFRs such as TNFR1. Collectively, this study provides more insights into RELT expression, RELT family member function, and the mechanism of RELT-induced death.

Keywords: Apoptosis; RELL1; RELL2; RELT; TNFR; p38.

Figure 1. RELT protein expression in human tissues

RELT expression was detected in human tissue extracts by western blotting as described in Materials and Methods. The blot was reprobed with an anti-GADPH primary antibody for a loading control (PBL, peripheral blood leukocyte).

Figure 2. RELT family members activate p38

A. HEK-293 cells were transfected with the indicated expression plasmids and assayed for p38 indirectly through measurement of CHOP mediated luciferase expression as described in Materials and Methods. Data are normalized to empty vector. Significant values of P<0.05 (*) and P<0.005 (**) are indicated. A. Transfection of empty vector plasmid or expression plasmids for RELT, OSR1, OSR1 K46M (KM), TRAF2, TRAF2 RING mutant (RING) and MEKK3 as indicated. B. Transfection of empty vector plasmid or co-transfection of expression plasmids for RELL1 (L1) with the indicated plasmid. C. Same as in B, except that co-transfection of expression plasmid for RELL2 (L2) with the indicated plasmid.

Figure 3. RELT deletion mutants

A. Map of deletion mutants of RELT constructed utilizing site-directed mutagenesis. The predicted ECD, TMD highlighted in black and ICD of each construct are indicated. Mutants E, F, G, H and I were designed to possess a truncated carboxyl-terminal end as indicated. Mutant B was designed to contain an internal Δ192-259 deletion and mutant D was designed to contain a Δ252-420 deletion. B. Western blot of RELT mutant expression. 293 cells were transfected with the indicated expression plasmids and protein lysates were analyzed by western blotting as described in Materials and Methods. Two images of the same representative blot are shown and overexposure of the representative blot is indicated. C. Ability of RELT mutants to induce apoptotic morphology. 293 cells were transfected with the indicated expression plasmids together with an expression plasmid for the β-galactosidase gene. Cells were stained with X-gal as described in Materials and Methods. Results are expressed as the percentage of β-galactosidase positive cells that are undergoing cell death based on morphology. Significant values of P<0.005 (**) are indicated. D. Ability of RELT mutants to induce DNA fragmentation. 293 cells were transfected with expression plasmid for GFP together with the indicated expression plasmid and assayed for DNA fragmentation as described in Materials and Methods. Results are expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are solely GFP positive. Significant values of P<0.05 (*) and P<0.005 (**) are indicated. E. Ability of RELT mutants to activate p38. 293 cells were either left untreated (UNT), treated with sorbitol for 1 or 2 hours (hr), or transfected with the indicated expression plasmids: empty vector (V), FL RELT, or for the indicated RELT deletion mutants. Protein lysates were collected and analyzed by western blotting for phosphorylated p-38 (top panel - red) as well as alpha and beta isoforms of p38 regardless of phosphorylated state (bottom panel - green) as described in Materials and Methods.

Figure 3. RELT deletion mutants

A. Map of deletion mutants of RELT constructed utilizing site-directed mutagenesis. The predicted ECD, TMD highlighted in black and ICD of each construct are indicated. Mutants E, F, G, H and I were designed to possess a truncated carboxyl-terminal end as indicated. Mutant B was designed to contain an internal Δ192-259 deletion and mutant D was designed to contain a Δ252-420 deletion. B. Western blot of RELT mutant expression. 293 cells were transfected with the indicated expression plasmids and protein lysates were analyzed by western blotting as described in Materials and Methods. Two images of the same representative blot are shown and overexposure of the representative blot is indicated. C. Ability of RELT mutants to induce apoptotic morphology. 293 cells were transfected with the indicated expression plasmids together with an expression plasmid for the β-galactosidase gene. Cells were stained with X-gal as described in Materials and Methods. Results are expressed as the percentage of β-galactosidase positive cells that are undergoing cell death based on morphology. Significant values of P<0.005 (**) are indicated. D. Ability of RELT mutants to induce DNA fragmentation. 293 cells were transfected with expression plasmid for GFP together with the indicated expression plasmid and assayed for DNA fragmentation as described in Materials and Methods. Results are expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are solely GFP positive. Significant values of P<0.05 (*) and P<0.005 (**) are indicated. E. Ability of RELT mutants to activate p38. 293 cells were either left untreated (UNT), treated with sorbitol for 1 or 2 hours (hr), or transfected with the indicated expression plasmids: empty vector (V), FL RELT, or for the indicated RELT deletion mutants. Protein lysates were collected and analyzed by western blotting for phosphorylated p-38 (top panel - red) as well as alpha and beta isoforms of p38 regardless of phosphorylated state (bottom panel - green) as described in Materials and Methods.

Figure 3. RELT deletion mutants

A. Map of deletion mutants of RELT constructed utilizing site-directed mutagenesis. The predicted ECD, TMD highlighted in black and ICD of each construct are indicated. Mutants E, F, G, H and I were designed to possess a truncated carboxyl-terminal end as indicated. Mutant B was designed to contain an internal Δ192-259 deletion and mutant D was designed to contain a Δ252-420 deletion. B. Western blot of RELT mutant expression. 293 cells were transfected with the indicated expression plasmids and protein lysates were analyzed by western blotting as described in Materials and Methods. Two images of the same representative blot are shown and overexposure of the representative blot is indicated. C. Ability of RELT mutants to induce apoptotic morphology. 293 cells were transfected with the indicated expression plasmids together with an expression plasmid for the β-galactosidase gene. Cells were stained with X-gal as described in Materials and Methods. Results are expressed as the percentage of β-galactosidase positive cells that are undergoing cell death based on morphology. Significant values of P<0.005 (**) are indicated. D. Ability of RELT mutants to induce DNA fragmentation. 293 cells were transfected with expression plasmid for GFP together with the indicated expression plasmid and assayed for DNA fragmentation as described in Materials and Methods. Results are expressed as the percentage of cells that are both TUNEL and GFP positive versus cells that are solely GFP positive. Significant values of P<0.05 (*) and P<0.005 (**) are indicated. E. Ability of RELT mutants to activate p38. 293 cells were either left untreated (UNT), treated with sorbitol for 1 or 2 hours (hr), or transfected with the indicated expression plasmids: empty vector (V), FL RELT, or for the indicated RELT deletion mutants. Protein lysates were collected and analyzed by western blotting for phosphorylated p-38 (top panel - red) as well as alpha and beta isoforms of p38 regardless of phosphorylated state (bottom panel - green) as described in Materials and Methods.

Figure 4. RELT-induced death is independent of FADD and Caspase-8

293 cells were transfected with RELT or TNFR1 in combination with the indicated inhibitor of apoptosis: CrmA, the Fadd dominant negative mutant (FaddDN) or a Caspase-8 C360S dominant negative mutant (C8 mutant). Additionally, each well was transfected with an expression plasmid for β-galactosidase. Cells were stained with X-gal 48 hours after transfection as described in Materials and Methods. Results are expressed as the percentage of β-galactosidase positive cells that are undergoing cell death based on morphology. Significant values of P<0.005 (**) are indicated. B. Representative bright-field images under x20 magnification of cells transfected with the indicated expression plasmids.