Kelch Repeat and BTB Domain Containing Protein 5 (Kbtbd5) Regulates Skeletal Muscle Myogenesis through the E2F1-DP1 Complex.

We have previously isolated a muscle-specific Kelch gene, Kelch repeat and BTB domain containing protein 5 (Kbtbd5)/Kelch-like protein 40 (Klhl40). In this report, we identified DP1 as a direct interacting factor for Kbtbd5 using a yeast two-hybrid screen and in vitro binding assays. Our studies demonstrate that Kbtbd5 interacts and regulates the cytoplasmic localization of DP1. GST pulldown assays demonstrate that the dimerization domain of DP1 interacts with all three of the Kbtbd5 domains. We further show that Kbtbd5 promotes the ubiquitination and degradation of DP1, thereby inhibiting E2F1-DP1 activity. To investigate the in vivo function of Kbtbd5, we used gene disruption technology and engineered Kbtbd5 null mice. Targeted deletion of Kbtbd5 resulted in postnatal lethality. Histological studies reveal that the Kbtbd5 null mice have smaller muscle fibers, a disorganized sarcomeric structure, increased extracellular matrix, and decreased numbers of mitochondria compared with wild-type controls. RNA sequencing and quantitative PCR analyses demonstrate the up-regulation of E2F1 target apoptotic genes (Bnip3 and p53inp1) in Kbtbd5 null skeletal muscle. Consistent with these observations, the cellular apoptosis in Kbtbd5 null mice was increased. Breeding of Kbtbd5 null mouse into the E2F1 null background rescues the lethal phenotype of the Kbtbd5 null mice but not the growth defect. The expression of Bnip3 and p53inp1 in Kbtbd5 mutant skeletal muscle are also restored to control levels in the E2F1 null background. In summary, our studies demonstrate that Kbtbd5 regulates skeletal muscle myogenesis through the regulation of E2F1-DP1 activity.

Kbtbd5 is regulated by MyoD and restricted to the myogenic lineage.

BTB-BACK-Kelch (BBK) proteins play broad roles in cellular and molecular regulation. The role of BBK proteins in the skeletal muscle lineage and myogenesis remains an active area of research. Herein, we report a novel BBK gene, Kbtbd5, which we discovered and found to be restricted to the myogenic lineage. We observed that Kbtbd5 was absent in proliferating myoblasts and upregulated upon myogenic differentiation. In situ hybridization analysis revealed that Kbtbd5 was restricted to the skeletal muscle lineage during embryogenesis. We identified a conserved 1.2kb upstream region, which directs reporter expression to the developing skeletal muscle lineage. Transcriptional and mutagenesis assays demonstrated that the E-box motifs contribute to the Kbtbd5 promoter activity. We have also demonstrated the in vivo and in vitro binding between MRFs and the E-box motif in the 1.2kb promoter of the Kbtbd5 gene. Our studies have revealed that the Myod family can transactivate the 1.2kb-luc reporter through the E-box motifs. In addition, we have shown that Kbtbd5 can recruit the Cullin 3 complex in vivo. Using shRNA knockdown, our study has revealed that Kbtbd5 plays an important role in the myogenic differentiation. In summary, we have demonstrated that Kbtbd5 is the direct downstream target gene of the Myod family and regulates myogenic differentiation. Our results further support the notion that Kbtbd5 may serve as an adapter of Cul3 during myogenic differentiation.

ER71 directs mesodermal fate decisions during embryogenesis.

Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.

Proteome of human calcium kidney stones.

OBJECTIVES: Idiopathic calcium oxalate (CaOx) stones are believed to develop attached to papillary subepithelial deposits called Randall's plaques. Calcium phosphate (CaP) stones, conversely, are thought to arise within the inner medullary collecting ducts, enlarging and damaging surround tubular structures as they expand. If this is true, we theorize that differences will be seen within the organic portion (matrix) of CaOx stones compared with CaP stones using a mass spectroscopy (MS) approach. METHODS: From a cohort of 47 powdered stones, 25 calculi (13 CaOx, 12 CaP) were confirmed to contain a dominant mineral content of >80% by powder x-ray diffraction. Matrix proteins were then extracted, purified, and digested. Peptide tandem MS data were acquired, and spectra were searched against a large human protein database to identify protein matches. RESULTS: No significant differences were seen between pattern profiles of CaOx and CaP stones. However, variations in protein expression patterns were seen within individual CaOx (monohydrate and dihydrate) and CaP (apatite and brushite) mineral subtypes, suggesting a relationship between crystal-surface binding properties and matrix composition. Both groups contain a large number of inflammatory proteins and a catalog of common proteins is included. CONCLUSIONS: Calcium kidney stone matrix contains hundreds of proteins and is predominated by proteins associated with inflammatory response. Many of the same proteins were identified in both CaOx and CaP stones, suggesting inflammation as a unifying origin or a common secondary role in calcium stone pathogenesis.

Fhl2 interacts with Foxk1 and corepresses Foxo4 activity in myogenic progenitors.

Adult skeletal muscle has a remarkable regenerative capacity because of a myogenic progenitor cell population. Using a gene disruption strategy, we determined that Foxk1 regulates myogenic progenitor cell activation and muscle regeneration. In this study, we undertook a yeast two hybrid screen to identify Foxk1 interacting proteins. We identified the LIM-only protein, Fhl2, as a Foxk1 interacting protein. Using transcriptional assays, we observed that Fhl2, in a dose-dependent fashion, promotes Foxk1 transcriptional repression of Foxo4 activity. Using histochemical and immunohistochemical assays, we further established that Fhl2 is expressed in the myogenic progenitor cell population. Fhl2 knockdown results in cell cycle arrest, and mice lacking Fhl2 have perturbed skeletal muscle regeneration. Collectively, these studies define a Fhl2-Foxk1 cascade that regulates the myogenic progenitor cell activity in adult skeletal muscle and enhances our understanding of muscle regeneration.

Lead placement and associated nerve distribution of an implantable periurethral electrostimulator.

The aim of this study was to determine gross and neuroanatomic features of a novel periurethral neuromuscular electrostimulator. Periurethral leads were placed in eight female cadavers. In two cases, leads were imaged after placement to enhance anatomic understanding. Pelvic viscera were removed en bloc for analysis of lead placement in the six remaining cadavers. Excised tissue was sectioned and immunostained to identify general, afferent, sympathetic, and nitric oxide synthase efferent nerve fibers. The electrodes were found within/lateral (n = 4), within/posterolateral (n = 9), and anterolateral (n = 1) to the external urethral sphincter (distance 0.25 +/- 0.5, 2.9 +/- 3.3, and 1.0 +/- 0.0 mm, respectively). The electrode to the urethra and vagina distance averaged 7.6 +/- 3.4 and 8.8 +/- 4.3 mm, respectively. Variable density staining for all nerve types was found around the electrode. A periurethral electrode interfaces the external urethral sphincter, and the adjacent distribution of nerve fibers supports proposed neuromuscular therapeutic mechanisms.

Association of the protein D and protein E forms of rat CRISP1 with epididymal sperm.

Cysteine-rich secretory protein 1 (CRISP1) is a secretory glycoprotein produced by the rat epididymal epithelium in two forms, referred to as proteins D and E. CRISP1 has been implicated in sperm-egg fusion and has been shown to suppress capacitation in rat sperm. Several studies have suggested that CRISP1 associates transiently with the sperm surface, whereas others have shown that at least a portion of CRISP1 persists on the surface. In the present study, we demonstrate that protein D associates transiently with the sperm surface in a concentration-dependent manner, exhibiting saturable binding to both caput and cauda sperm in a concentration range that is consistent with its capacitation-inhibiting activity. In contrast, protein E persists on the sperm surface after all exogenous protein D has been dissociated. Comparison of caput and cauda sperm reveal that protein E becomes bound to the sperm in the cauda epididymidis. We show that protein E associates with caput sperm, which do not normally have it on their surfaces, in vitro in a time- and temperature-dependent manner. These studies demonstrate that most CRISP1 interacts with sperm transiently, possibly with a specific receptor on the sperm surface, consistent with its action in suppressing capacitation during epididymal storage of sperm. These studies also confirm a tightly bound population of protein E that could act in the female tract.

Structure and function of epididymal protein cysteine-rich secretory protein-1.

Cysteine-rich secretory protein-1 (CRISP-1) is a glycoprotein secreted by the epididymal epithelium. It is a member of a large family of proteins characterized by two conserved domains and a set of 16 conserved cysteine residues. In mammals, CRISP-1 inhibits sperm-egg fusion and can suppress sperm capacitation. The molecular mechanism of action of the mammalian CRISP proteins remains unknown, but certain non-mammalian CRISP proteins can block ion channels. In the rat, CRISP-1 comprises two forms referred to as Proteins D and E. Recent work in our laboratory demonstrates that the D form of CRISP-1 associates transiently with the sperm surface, whereas the E form binds tightly. When the spermatozoa are washed, the E form of CRISP-1 persists on the sperm surface after all D form has dissociated. Cross-linking studies demonstrate different protein-protein interaction patterns for D and E, although no binding partners for either protein have yet been identified. Mass spectrometric analyses revealed a potential post-translational modification on the E form that is not present on the D form. This is the only discernable difference between Proteins D and E, and presumably is responsible for the difference in behavior of these two forms of rat CRISP-1. These studies demonstrate that the more abundant D form interacts with spermatozoa transiently, possibly with a specific receptor on the sperm surface, consistent with a capacitation-suppressing function during sperm transit and storage in the epididymis, and also confirm a tightly bound population of the E form that could act in the female reproductive tract.