Tropomodulin 1 expression driven by NF-κB enhances breast cancer growth.

Triple-negative breast cancers (TNBC), which include the basal-like and claudin-low disease subtypes, are aggressive malignancies for which effective therapeutic targets are lacking. NF-κB activation has an established role in breast malignancy, and it is higher in TNBC than other breast cancer subtypes. On this basis, we hypothesized that proteins derived from NF-κB target genes might be molecular targets for TNBC therapy. In this study, we conducted a microarray-based screen for novel NF-κB-inducible proteins as candidate therapeutic targets, identifying tropomodulin 1 (TMOD1) as a lead candidate. TMOD1 expression was regulated directly by NF-κB and was significantly higher in TNBC than other breast cancer subtypes. TMOD1 elevation is associated with enhanced tumor growth in a mouse tumor xenograft model and in a 3D type I collagen culture. TMOD1-dependent tumor growth was correlated with MMP13 induction, which was mediated by TMOD1-dependent accumulation of ß-catenin. Overall, our study highlighted a novel TMOD1-mediated link between NF-κB activation and MMP13 induction, which accounts in part for the NF-κB-dependent malignant phenotype of TNBC.

Leiomodin 3 and tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis.

Precise regulation of thin filament length is essential for optimal force generation during muscle contraction. The thin filament capping protein tropomodulin (Tmod) contributes to thin filament length uniformity by regulating elongation and depolymerization at thin filament ends. The leiomodins (Lmod1-3) are structurally related to Tmod1-4 and also localize to actin filament pointed ends, but in vitro biochemical studies indicate that Lmods act instead as robust nucleators. Here, we examined the roles of Tmod4 and Lmod3 during Xenopus skeletal myofibrillogenesis. Loss of Tmod4 or Lmod3 resulted in severe disruption of sarcomere assembly and impaired embryonic movement. Remarkably, when Tmod4-deficient embryos were supplemented with additional Lmod3, and Lmod3-deficient embryos were supplemented with additional Tmod4, sarcomere assembly was rescued and embryonic locomotion improved. These results demonstrate for the first time that appropriate levels of both Tmod4 and Lmod3 are required for embryonic myofibrillogenesis and, unexpectedly, both proteins can function redundantly during in vivo skeletal muscle thin filament assembly. Furthermore, these studies demonstrate the value of Xenopus for the analysis of contractile protein function during de novo myofibril assembly.

Leucine 135 of tropomodulin-1 regulates its association with tropomyosin, its cellular localization, and the integrity of sarcomeres.

Tropomodulin-1 (Tmod-1) is a well defined actin-capping protein that interacts with tropomyosin (TM) at the pointed end of actin filaments. Previous studies by others have mapped its TM-binding domain to the amino terminus from amino acid 39 to 138. In this study, we have identified several amino acid residues on Tmod-1 that are important for its interaction with TM5 (a nonmuscle TM isoform). Glutathione S-transferase affinity chromatography and immunoprecipitation assays reveal that Tmod sense mutations of either amino acid 134, 135, or 136 causes various degrees of loss of function of Tmod TM-binding ability. The reduction of TM-binding ability was relatively mild (reduced approximately 20-40%) from the G136A Tmod mutant but more substantially (reduced approximately 50-100%) from the I134D, L135E, and L135V Tmod mutants. In addition, mutation at any of these three sites dramatically alters the subcellular location of Tmod-1 when introduced into mammalian cells. Further analysis of these three mutants uncovered a previously unknown nuclear trafficking function of Tmod-1, and residues 134, 135, and 136 are located within a nuclear export signal motif. As a result, mutation on either residue 134 or residue 135 not only will cause a significant reduction of the Tmod-1 ability to bind to TM5 but also lead to predominant nuclear localization of Tmod-1 by crippling its nuclear export mechanism. The failure of the Tmod mutations to fully associate with TM5 when introduced into neonatal rat cardiomyocytes was also associated with an accelerated and severe fragmentation of sarcomeric structures compared with overexpression of wild type Tmod-1. The multiple losses of function of Tmod engendered by these missense mutations are most severe with the single substitution of residue 135.

Tropomodulin expression in developing hearts of normal and cardiac mutant Mexican axolotl.

In the axolotl, Ambystoma mexicanum, a simple, recessive cardiac-lethal mutation in gene "c" results in the hearts of c/c homozygous animals being deficient in sarcomeric tropomyosin (TM) and failing to form mature myofibrils. Subsequently, the mutant hearts do not beat. A three-step model of myofibril assembly recently developed in cell culture prompted a reassessment of the myofibril assembly process in mutant hearts using a relatively new late marker for thin filament assembly, tropomodulin (Tmod). This is, to the best of our knowledge, the first report of tropomodulin in an amphibian system. Tropomodulin antibodies were immunolocalized to the ends of the thin filaments. Tropomodulin was also found in discrete punctate spots in normal and mutant hearts, often in linear arrays suggestive of early myofibril formation. The tropomodulin spots assessed in stage 41/42 mutant hearts co-localized with antibodies to other myofibrillar proteins indicative of nascent myofibril formation. This suggests a failure of elongation/maturation of nascent myofibrils, which could be a consequence of decreased TM levels or increased Tmod/ TM ratio. Unlike tropomyosin, there is no apparent decrease in the level of Tmod expression in mutant hearts.