Valvular dystrophy associated filamin A mutations reveal a new role of its first repeats in small-GTPase regulation.

Filamin A (FlnA) is a ubiquitous actin binding protein which anchors various transmembrane proteins to the cell cytoskeleton and provides a scaffold to many cytoplasmic signaling proteins involved in actin cytoskeleton remodeling in response to mechanical stress and cytokines stimulation. Although the vast majority of FlnA binding partners interact with the carboxy-terminal immunoglobulin like (Igl) repeats of FlnA, little is known on the role of the amino-N-terminal repeats. Here, using cardiac mitral valvular dystrophy associated FlnA-G288R and P637Q mutations located in the N-terminal Igl repeat 1 and 4 respectively as a model, we identified a new role of FlnA N-terminal repeats in small Rho-GTPases regulation. Using FlnA-deficient melanoma and HT1080 cell lines as expression systems we showed that FlnA mutations reduce cell spreading and migration capacities. Furthermore, we defined a signaling network in which FlnA mutations alter the balance between RhoA and Rac1 GTPases activities in favor of RhoA and provided evidences for a role of the Rac1 specific GTPase activating protein FilGAP in this process. Together our work ascribed a new role to the N-terminal repeats of FlnA in Small GTPases regulation and supports a conceptual framework for the role of FlnA mutations in cardiac valve diseases centered around signaling molecules regulating cellular actin cytoskeleton in response to mechanical stress.

Periostin is expressed by cells of the human and sand rat intervertebral discs.

Periostin, a matricellular protein in the fasciclin family, is expressed in tissues subjected to constant mechanical stress. Periostin modulates cell-to-extracellular matrix interactions and can bind to collagen, fibronectin, tenascin-C and several integrins. Our objective was to evaluate whether periostin is expressed in the human intervertebral disc. Immunohistochemical localization of periostin was carried out in tissue of human lumbar discs and lumbar discs of the sand rat (Psammomys obesus). Human discs also were examined for periostin gene expression. Immunohistochemical localization demonstrated periostin in the cytoplasm of annulus and nucleus cells, and occasionally in the surrounding pericellular and interterritorial extracellular matrix. Periostin distribution in the human disc was distinctive. Outer annulus contained the highest proportion of periostin-positive cells (88.8%), whereas inner annulus contained only 61.4%. The nucleus pulposus contained the fewest periostin-positive cells (18.5%). There was a significant negative correlation between the percentage of cells positive for periostin in the inner annulus and subject age. Periostin gene expression in the human disc also was confirmed using molecular microarray analysis. Because work by others has shown that periostin plays an important role in the biomechanical properties of other connective tissues (skin, tendon, heart valves), future research is needed to elucidate the role of periostin in disc, loading, aging and degeneration.

Expression of the familial cardiac valvular dystrophy gene, filamin-A, during heart morphogenesis.

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

The identification of Prx1 transcription regulatory domains provides a mechanism for unequal compensation by the Prx1 and Prx2 loci.

Transcription regulatory domains of the Prx1a and Prx1b homeoproteins were analyzed in transient transfection assays using artificial promoters as well as an established downstream target promoter (tenascin-c). Activation and repression domains were detected in their common amino end. In the carboxyl end of Prx1a an activation domain and an inhibition/masking region (OAR domain) were detected. The Prx1b isoform, generated by alternative splicing, does not contain these carboxyl activation or inhibition domains. Instead, the data demonstrate that the carboxyl tail of Prx1b contains a potent repressor region. This difference in the carboxyl tail accounts for a 45-fold difference observed in transcription regulatory activity between Prx1a and Prx1b. The data also support the likelihood that this difference between Prx1a and Prx1b is higher in the presence of still undetermined cofactors. DNA binding affinities of Prx1a, Prx1b, and a series of truncation mutants were also examined. The carboxyl tail of Prx1a, which inhibited transcription activation in the transfection assays, also inhibited DNA binding. These differences in biochemical function between Prx1a and Prx1b, as well as the recently described activities of Prx2, provide a mechanism for the unequal compensation between the Prx1 and Prx2 loci.

Identification of domains mediating transcription activation, repression, and inhibition in the paired-related homeobox protein, Prx2 (S8).

Despite the growing information concerning the developmental importance of the Prx2 protein, the structural determinants of Prx2 function are poorly understood. To gain insight into the transcription regulatory regions of the Prx2 protein, we generated a series of truncation mutants. Both the Prx2 response element (PRE) and a portion of the tenascin promoter, a downstream target of Prx2, were used as reporters in transient transfection assays. This analysis showed that a conserved domain (PRX), found in both Prx1 and Prx2, activated transcription in NIH 3T3 cells. This PRX domain, as well as other functional regions of Prx2, demonstrated both cell-specific and promoter-dependent transcriptional regulation. A second important region, the OAR (aristaless) domain, which is conserved among 35 Paired-type homeodomain proteins, was observed to inhibit transcription. Deletion of this element resulted in a 20-fold increase of transcription from the tenascin reporter in NIH 3T3 cells but not in C2C12 cells. The OAR domain did not function as a repressor in chimeric fusions with the Gal4 DNA binding domain in either cell type, characterizing it as an inhibitor instead of a repressor. These results give insight into the function of the Prx2 transcription factor while establishing the framework for comparison with the two isoforms of Prx1.

The outflow tract of the heart is recruited from a novel heart-forming field.

As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease.

Human PRRX1 and PRRX2 genes: cloning, expression, genomic localization, and exclusion as disease genes for Nager syndrome.

In this study, we extend our examination of the function of the Prrx1 (a.k.a Mhox, Prx1, K-2, and Pmx1) as well as Prrx2 (a.k.a. S8 and Prx2) genes by characterizing the expression of the human orthologs and their potential for causing specific human malformations. The expression pattern of PRRX2 and its close relative, PRRX1, were analyzed in human tissue by RT-PCR. Although the expression of these human genes is similar to their mouse orthologs, there are notable differences in expression. PRRX2 was detected in the human kidney and lung, whereas in mice and chickens neither of these tissues has been reported to express Prrx2. For PRRX1 the expression pattern was quite similar to other vertebrates, but the ratio of the two isoforms was reversed. To begin the search for the gene-disease connection, both genes were mapped to human chromosomes by FISH. The PRRX1 locus maps to 1q23, whereas the PRRX2 locus maps to 9q34.1. This localization, along with the recently described phenotypes of the gene-targeted Prrx1, Prrx2 and double mutant mice, enabled us to search the human disease databases for similar malformations. This examination suggested that mutations at the PRRX1 and/or PRRX2 loci could result in Nager Acrofacial Dysostosis (NAFD) syndrome. We obtained DNA samples from eight patients with NAFD, as well as two patients with Miller syndrome, and analyzed them for mutations in the PRRX1 and PRRX2 genes. The data excludes mutations in the presumed coding sequences of these genes from causing NAFD.

Grasping the handrails during treadmill walking does not alter sagittal plane kinematics of walking.

OBJECTIVE: To determine whether grasping the handrails during treadmill walking affects sagittal plane kinematic parameters selected to describe walking style. DESIGN: Crossover trial. SETTING: A university motion analysis laboratory. PARTICIPANTS: A convenience sample comprised of 15 apparently healthy college-age volunteers. INTERVENTION: After being acclimatized to treadmill walking, subjects were videotaped while completing two treadmill walking bouts. Each bout was 10 minutes in duration and was conducted at a walking speed of 1.5m/sec. Subjects were instructed to grasp the handrails in one bout (GRASP) but to refrain from using the handrails in the other (FREE). Both bouts were conducted in a single session and were separated by a 10-minute rest period. The order in which subjects completed the bouts was randomized. MAIN OUTCOME MEASURES: Five successive strides occurring during the last 30 seconds of each bout were digitized. The coordinate data were numerically filtered and the following parameters derived: stride length, percentage of stride cycle spent in double-support, and the hip, knee, and ankle angles at heel-strike and toe-off. The results for the five strides in each bout were averaged and the average value was used in the statistical analysis. The FREE and GRASP conditions were compared with t tests for dependent samples (p < or = .05). RESULTS: There were no differences between the FREE and GRASP conditions for any of the parameters assessed. CONCLUSIONS: Subjects may grasp the treadmill handrails without affecting sagittal plane kinematic parameters of walking style.