Jeffrey A. Spencer

Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.

Elastic fibers are components of the extracellular matrix and confer resilience. Once laid down, they are thought to remain stable, except in the uterine tract where cycles of active remodeling occur. Loss of elastic fibers underlies connective tissue aging and important diseases including emphysema. Failure to maintain elastic fibers is explained by a theory of antielastase-elastase imbalance, but little is known about the role of renewal. Here we show that mice lacking the protein lysyl oxidase–like 1 (LOXL1) do not deposit normal elastic fibers in the uterine tract post partum and develop pelvic organ prolapse, enlarged airspaces of the lung, loose skin and vascular abnormalities with concomitant tropoelastin accumulation. Distinct from the prototypic lysyl oxidase (LOX), LOXL1 localizes specifically to sites of elastogenesis and interacts with fibulin-5. Thus elastin polymer deposition is a crucial aspect of elastic fiber maintenance and is dependent on LOXL1, which serves both as a cross-linking enzyme and an element of the scaffold to ensure spatially defined deposition of elastin.

Myosin accumulation and striated muscle myopathy result from the loss of muscle RING finger 1 and 3

Maintenance of skeletal and cardiac muscle structure and function requires precise control of the synthesis, assembly, and turnover of contractile proteins of the sarcomere. Abnormalities in accumulation of sarcomere proteins are responsible for a variety of myopathies. However, the mechanisms that mediate turnover of these long-lived proteins remain poorly defined. We show that muscle RING finger 1 (MuRF1) and MuRF3 act as E3 ubiquitin ligases that cooperate with the E2 ubiquitin-conjugating enzymes UbcH5a, -b, and -c to mediate the degradation of beta/slow myosin heavy chain (beta/slow MHC) and MHCIIa via the ubiquitin proteasome system (UPS) in vivo and in vitro. Accordingly, mice deficient for MuRF1 and MuRF3 develop a skeletal muscle myopathy and hypertrophic cardiomyopathy characterized by subsarcolemmal MHC accumulation, myofiber fragmentation, and diminished muscle performance. These findings identify MuRF1 and MuRF3 as key E3 ubiquitin ligases for the UPS-dependent turnover of sarcomeric proteins and reveal a potential basis for myosin storage myopathies.

Loss of muscle-specific RING-finger 3 predisposes the heart to cardiac rupture after myocardial infarction

RING-finger proteins commonly function as ubiquitin ligases that mediate protein degradation by the ubiquitin-proteasome pathway. Muscle-specific RING-finger (MuRF) proteins are striated muscle-restricted components of the sarcomere that are thought to possess ubiquitin ligase activity. We show that mice lacking MuRF3 display normal cardiac function but are prone to cardiac rupture after acute myocardial infarction. Cardiac rupture is preceded by left ventricular dilation and a severe decrease in cardiac contractility accompanied by myocyte degeneration. Yeast two-hybrid assays revealed four-and-a-half LIM domain (FHL2) and γ-filamin proteins as MuRF3 interaction partners, and biochemical analyses showed these proteins to be targets for degradation by MuRF3. Accordingly, FHL2 and γ-filamin accumulated to abnormal levels in the hearts of mice lacking MuRF3. These findings reveal an important role of MuRF3 in maintaining cardiac integrity and function after acute myocardial infarction and suggest that turnover of FHL2 and γ-filamin contributes to this cardioprotective function of MuRF3.

Alteration of mesodermal cell differentiation by EWS/FLI-1, the oncogene implicated in Ewing's sarcoma.

ABSTRACT The chimeric fusion gene EWS/FLI-1 is detected in most cases of Ewings sarcoma (ES), the second most common malignant bone tumor of childhood. Although 80% of ES tumors develop in skeletal sites, the remainder can arise in almost any soft tissue location. The lineage of the cell developing the EWS/FLI-1 gene fusion has not been fully characterized but is generally considered to be of either mesenchymal or neural crest origin. To study this oncogene in a conceptually relevant target cell, EWS/FLI-1 was introduced into the murine cell line C2C12, a myoblast cell line capable of differentiation into muscle, bone, or fat. In this cellular context, EWS/FLI-1 profoundly inhibited the myogenic differentiation program. The block in C2C12 myogenic differentiation required the nuclear localization and DNA-binding functions of EWS/FLI-1 and was mediated by transcriptional and posttranscriptional suppression of the myogenic transcription factors MyoD and myogenin. Interestingly, C2C12-EWS/FLI-1 cells constitutively expressed alkaline phosphatase, a bone lineage marker, and were alkaline phosphatase positive by histochemistry but showed no other evidence of bone lineage commitment. Consistent with recent findings in human ES tumor cell lines, C2C12-EWS/FLI-1 cells constitutively expressed cyclin D1 and demonstrated decreased expression of the cell cycle regulator p21cip1, even under differentiation conditions and at confluent density. This C2C12-EWS/FLI-1 cell model may assist in the identification of novel differentially expressed genes relevant to ES and provide further insight into the cell(s) of origin developing ES-associated genetic fusions.

Cooperative Transcriptional Activation by Serum Response Factor and the High Mobility Group Protein SSRP1

Serum response factor (SRF) is a MADS box transcription factor that controls a wide range of genes involved in cell proliferation and differentiation. The MADS box mediates homodimerization and binding of SRF to the consensus sequence CC(A/T)6GG, known as a CArG box, which is found in the control regions of numerous serum-inducible and muscle-specific genes. Using a modified yeast one-hybrid screen to identify potential SRF cofactors, we found that SRF interacts with the high mobility group factor SSRP1 (structure-specific recognition protein). This interaction, which occurs in yeast and mammalian cells, is mediated through the MADS box of SRF and a basic region of SSRP1 encompassing amino acids 489–542, immediately adjacent to the high mobility group domain. SSRP1 does not bind the CArG box, but interaction of SSRP1 with SRF dramatically increases the DNA binding activity of SRF, resulting in synergistic transcriptional activation of native and artificial SRF-dependent promoters. These results reveal an important role for SSRP1 as a coregulator of SRF-dependent transcription in mammalian cells.

Selective identification of secreted and transmembrane breast cancer markers using Escherichia coli ampicillin secretion trap.

Secreted and cell surface proteins play important roles in cancer and are potential drug targets and tumor markers. Here, we describe a large-scale analysis of the genes encoding secreted and cell surface proteins in breast cancer. To identify these genes, we developed a novel signal sequence trap method called Escherichia coli ampicillin secretion trap (CAST). For CAST, we constructed a plasmid in which the signal sequence of beta-lactamase was deleted such that it does not confer ampicillin resistance. Eukaryotic cDNA libraries cloned into pCAST produced tens of thousands of ampicillin-resistant clones, 80% of which contained cDNA fragments encoding secreted and membrane spanning proteins. We identified 2,708 unique sequences from cDNA libraries made from surgical breast cancer specimens. We analyzed the expression of 1,287 of the 2,708 genes and found that 166 were overexpressed in breast cancers relative to normal breast tissues. Eighty-five percent of these genes had not been previously identified as markers of breast cancer. Twenty-three of the 166 genes (14%) were relatively tissue restricted, suggesting use as cancer-specific targets. We also identified several new markers of ovarian cancer. Our results indicate that CAST is a robust, rapid, and low cost method to identify cell surface and secreted proteins and is applicable to a variety of relevant biological questions.