TRAM2 Protein Interacts with Endoplasmic Reticulum Ca²⁺ Pump Serca2b and Is Necessary for Collagen Type I Synthesis

Abstract

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50-to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca 2؉ pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the Cterminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca 2؉ with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca 2؉ concentration at the site of collagen synthesis, and a high Ca 2؉ concentration may be necessary for the function of molecular chaperones involved in collagen folding.

Intracellular Calreticulin Regulates Multiple Steps in Fibrillar Collagen Expression, Trafficking, and Processing into the Extracellular Matrix

“…Studies suggest that high ER Ca 2ϩ levels are important for the proper folding of collagen (51). Furthermore, the interaction between CRT and collagen might be Ca 2ϩ -dependent; the conformation of CRT is Ca 2ϩ -sensitive, and this has been shown to affect CRT binding to other chaperones and proteins in the ER (65,66).…”

“…Therefore, these data suggest that intracellular CRT, rather than extracellular or cell surface-bound CRT, is primarily involved in regulating collagen expression by these fibroblasts. concentration in the ER has been suggested to be necessary for the proper chaperoning of collagen (51). To determine whether CRT regulation of ER Ca 2ϩ release is involved in regulation of collagen expression in these cells, wild type and CRT Ϫ/Ϫ MEFs were treated for 2 h with 100 nM thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase, which decreases ER Ca 2ϩ stores available for release.…”

Mechanisms of Hepatic Fibrogenesis

“…121 Fibrogenesis-Collagen type I is the prototype constituent of the fibril-forming matrix in fibrotic liver, and its expression is regulated both transcriptionally and posttranscriptionally as described earlier and in several reviews. [122][123][124] TGFβ1, derived from both paracrine and autocrine sources, remains the classic fibrogenic cytokine (see Inagaki and Okazaki 124 and Breitkopf et al 125 for reviews). Signals downstream of TGFβ converge on Smad proteins, which fine-tune and enhance the effects of TGFβ during stellate cell activation; Smads 2 and 3 are stimulatory whereas Smad 7 is inhibitory 124,126,127 and is antagonized by Id1.…”

Molecular Hallmarks of Endogenous Chromatin Complexes Containing Master Regulators of Hematopoiesis

“…7B, right), as expected (26), and also repressed Sox6, Tram2, and Etv6 (9.1-, 21-, and 3.2-fold, respectively). Tram2 is a BMP and Runx2 target gene in osteoblasts (67) and regulates protein translocation in the endoplasmic reticulum (75). Etv6 (Tel), a member of the Ets transcription factor family, is a key regulator of adult HSCs that is frequently disrupted via leukemogenic chromosomal translocations (30,93).…”