PMC

Sequence comparison between human TRAM, TRAM2, and BC030831. The amino acid sequences of TRAM (accession no. S30034), TRAM2 (KIAA0057; accession no. D31762), and TRAM-like protein (accession no. BC030831) were aligned using the ClustalW program. Identities are indicated as asterisks under the sequence, conserved residues in at least two proteins are shown as double dots, and conservative substitutions are indicated by single dots. Putative transmembrane regions are overlined with dotted lines, and the most divergent C-terminal domain is underlined with a solid line.

Phenotype of rat HSCs expressing C-terminus-truncated TRAM2 (TRAM2ΔC). Rat HSCs were infected at day 2 after isolation with adenoviruses expressing TRAM2 with deletion of 66 amino acids at the C terminus (top left panel), expressing full-size human TRAM2 (top right panel), or expressing control virus (CON, bottom left panel). All viruses also expressed GFP as a marker of infection. After an additional 5 days (7 days after isolation), an image of the cells was taken under UV light so that only cells expressing the viruses were visible. In the bottom right panel are HSCs cultured without virus for the same time period. This image was taken under visible light.

TRAM2ΔC overexpression inhibits pro-α1(I) in rat HSCs. (A) Decrease in the cellular level of pro-α1(I). Rat HSCs were infected with adenovirus expressing TRAM2ΔC (lane 1) or control adenovirus (CON, lane 2) at day 2 after isolation and cultured for an additional 5 days. Western blots with 50 μg of cellular proteins were probed under reducing conditions with antibodies specific for α-SMA and pro-α1(I). Probing with anti-HA antibody (HA) was done as a control for TRAM2ΔC expression, and probing with anti-GFP antibody (GFP) served as a control for viral infection. (B) TRAM2 overexpression does not affect the level of pro-α1(I) in HSCs. This experiment was performed similar to that shown in panel A, except adenoviruses expressing full-size TRAM2 (TRAM2, lane 2) and control virus (CON, lane 1) were used. Western blotting was done with anticollagen antibody [pro-α1(I)] or anti-HA antibody (HA) to verify TRAM2 expression. (C) Two samples of cell medium of cells expressing TRAM2ΔC (lanes 1 and 2) or cells expressing control virus (lanes 3 and 4) were analyzed by Western blotting as described for panel A. Migration of the pro-α1(I) band is indicated.

TRAM2 and Serca2b proteins interact in human fibroblasts. (A) TRAM2 coprecipitates with Serca2b and with type I procollagen. FLAG-tagged human Serca2b protein and HA-tagged human TRAM2 protein were expressed in human fibroblasts. Immunoprecipitation (IPP) was done with 1 mg of cellular extract and using anti-FLAG antibody (lane 2; FL), antitubulin antibody (lane 3; TUB), or anticollagen antibody (lane 4; COLL). Western blotting (WEST) was done with the precipitated material using anti-HA antibody (HA). Lane 1 is Western blotting only, showing expression of HA-TRAM2 in 50 μg of cellular extract. (B) TRAM2ΔC does not interact with Serca2b. This experiment was done as described for that in panel A, except that HA-tagged TRAM2ΔC and FLAG-tagged Serca2b were coexpressed. Immunoprecipitation (IPP) was done with anti-FLAG antibody (lane 2) or anti-HA antibody (lane 3), and Western blotting (WEST) was done with the precipitated material using anti-HA antibody (lanes 2 and 3). Lane 1 is Western blotting only, showing expression of Serca2b in 50 μg of cell extract. (C) Serca2b coprecipitates with TRAM2, but not with procollagen. Immunoprecipitations were done with the same material as used for panel A, except that antibodies used for IPP were anti-HA (lane 2, HA) or anticollagen (lane 3, COLL). Western blotting was done with anti-FLAG antibody (FLAG). Lane 1 is Western blotting only, showing expression of FLAG-Serca2b in 50 μg of cellular extract. (D) Calnexin does not coprecipitate with TRAM2. Immunoprecipitations were done with the same material as used for panel A, except that the antibody used for IPP was anti-HA (lane 2, HA). Western blotting was done with anticalnexin antibody (CLNX). Lane 1 is Western blotting only, showing expression of calnexin in 50 μg of cellular extract. (E) Interaction of TRAM2 and procollagen is translation dependent. Cells were incubated with puromycin (+) or without (−) for 2 h, and extracts were analyzed by Western blotting for expression of procollagen and TRAM2 (lanes 1 and 2). The same samples were subjected to immunoprecipitation with anticollagen antibody (COLL) (lanes 3 and 4). Western blotting was done with anti-HA antibody (HA). The signal from coimmunoprecipitated TRAM2 protein in the absence (−) or presence (+) of puromycin is indicated as TRAM2.

TRAM2 mRNA is induced in activated HSCs. (A) Expression in rat HSCs. Shown are results of RT-PCR analysis of RNA isolated from fresh rat HSCs (Q) or from rat HSCs activated in culture for 7 days (A). Twenty nanograms of poly(A)⁺ RNA (lanes 1 and 2) or 100 ng of total RNA (lanes 3 and 4) from two independent isolates of HSCs was subjected to RT-PCR analysis with primers specific for rat TRAM2. Poly(A)⁺ RNA samples were also analyzed with primers specific to GAPDH (bottom panel). Migration of TRAM2-specific bands is indicated. Both bands represent the same PCR product, as confirmed by sequencing (see Results). (B) Expression of TRAM and Serca2b in rat HSCs. RT-PCR was done as described for panel A, with 100 ng of total RNA extracted from quiescent (Q) and activated (A) rat HSCs and with primers specific for TRAM (top panel), Serca2b (middle panel), or GAPDH (bottom panel). Migration of PCR products is indicated. GAPDH served as an internal control. (C) Expression in human HSCs. A 100-ng aliquot of total RNA from freshly isolated human HSCs (0, lane 1) or from human HSCs cultured for 2 days (lane 2), 12 days (lane 3), or 17 days (lane 4) was analyzed by RT-PCR with primers specific for human TRAM2. Identity of the bands was confirmed by sequencing and is indicated on the right.

Inhibition of Serca2b inhibits disulfide bonding and intracellular degradation of pro-α1(I). (A) Thapsigargin causes a transient decrease in the cellular pro-α1(I) steady-state level. Rat fibroblasts were treated with thapsigargin (lanes 1 to 4) or DMSO (lanes 5 to 7) for the indicated time periods. Cellular proteins were analyzed by Western blotting with pro-α1(I)-specific antibody under reducing con-ditions. (B) Thapsigargin does not affect the steady-state level of collagen α1(I) mRNA. From the same cells as used in panel A, total RNA was extracted and 10 μg was analyzed by RNase protection assay with riboprobe specific to collagen α1(I) mRNA and GAPDH mRNA, as a control. Migration of the specific protected bands is indicated. (C) The proteosome inhibitor MG132 blocked the decrease in intracellular pro-α1(I). Fibroblasts were incubated with DMSO (lane 1), with 200 nM thapsigargin (lane 2), with 100 μM MG132 (lane 3), or with both thapsigargin and MG132 (lane 4) for 6 h. Cellular proteins were extracted and analyzed by Western blotting for pro-α1(I) (top panel) and α-tubulin (bottom panel) under reducing conditions. (D) The proteosome inhibitor lactacystin blocks the thapsigargin-induced decrease in intracellular pro-α1(I). The experiment was done as described for panel C, except that lactacystin was added at 50 μM. Only expression of pro-α1(I) is shown. (E) Thapsigargin completely inhibits formation of disulfide-bonded procollagen. Fibroblasts were incubated with thapsigargin (lanes 1 to 3) for the indicated time periods. Lane 4 represents control cells incubated with DMSO for 12 h. Cellular proteins were extracted, and 50 μg was analyzed by Western blotting under nonreducing conditions with pro-α1(I)-specific antibody. Migration of high-molecular-weight DBC and pro-α1(I) monomers is indicated on the left.

Inhibition of Serca2b decreases pro-α1(I) synthesis. (A) Thapsigargin depletes Ca²⁺ from the ER. A 200 nM concentration of thapsigargin was added to rat fibroblasts at time zero, and the increase in cytoplasmic Ca⁺ concentration was monitored by changes in Fluo-4 fluorescence. Control cells received DMSO. Cells were incubated in Ca²⁺-free medium, which caused a decrease in cytoplasmic Ca²⁺ at later time points. (B) Thapsigargin decreases the intracellular level of pro-α1(I). Western blotting results with cellular proteins from HSCs treated with 200 nM thapsigargin (lanes 1 to 3) or treated withDMSO (lanes 4 and 5) for the indicated time periods are shown. The blot was probed with anti-pro-α1(I) antibody (top panel) and anti-α-tubulin antibody (bottom panel). A 50-μg aliquot of protein was analyzed under reducing conditions. (C) Secretion of pro-α1(I) into cellular medium of HSCs is blocked by thapsigargin. At time zero cell medium was changed and thapsigargin was added to the cells (lanes 1 to 3), or DMSO was added (lanes 4 to 5). At the indicated time points, an aliquot of the medium was collected and analyzed by Western blotting with pro-α1(I)-specific antibody under reducing conditions. Migration of pro-α1(I) is indicated to the left. (D) The fibronectin level was unaffected by thapsigargin treatment. Cells were treated as described for panel B, and the cellular level of fibronectin was determined by Western blotting (upper panel). The same samples were probed for α-tubulin (lower panel), as a control for loading. (E) Calmidazolium Cl decreases pro-α1(I). The experiment was done as described for that in panel B, except calmidazolium Cl was added at 10 μM. One additional time point was taken at 6 h. Western blotting was done with anticollagen antibody [pro-α1(I)].