Citations

AbstractThe IGF-binding protein-4 (IGFBP-4) protease system is an important regulator of local IGF bioavailability and cell growth. Recently, the IGF-dependent IGFBP-4 protease secreted by cultured human fibroblasts was identified as pregnancy-associated plasma protein A (PAPP-A). In pregnancy serum, PAPP-A circulates as a disulfide-bound complex with the precursor form of major basic protein (pro-MBP), and in this complex PAPP-A’s proteolytic activity is not evident. In this study we analyzed the IGFBP-4 protease system in normal human fibroblasts to determine regulation outside of pregnancy. Treatment with the phorbol ester tumor promoter, β-phorbol 12,13-didecanoate (β-PDD), resulted in time-dependent inhibition of the IGF-dependent IGFBP-4 protease activity in cell-conditioned medium, which was evident at 6 h and complete by 24 h. PAPP-A mRNA was constitutively expressed in control cells, and levels were decreased only after 24 h of β-PDD treatment. Secretion of PAPP-A protein into conditioned medium did not change with β-PDD treatment. On the other hand, pro-MBP mRNA was undetectable in control human fibroblasts, and treatment with β-PDD induced pro-MBP mRNA and protein expression within 6 h. β-PDD-induced pro-MBP mRNA expression and protease inhibition were blocked with an inhibitor of RNA synthesis, actinomycin D. Actinomycin D had no effect on PAPP-A mRNA levels in the absence or presence of β-PDD. Similarly, transformation of human fibroblasts with simian virus 40 large T antigen resulted in the synthesis of pro-MBP mRNA and protein and inhibition of IGFBP-4 protease activity. Coculture of fibroblasts with cells transfected with pro-MBP cDNA resulted in inhibition of IGFBP-4 proteolytic activity without having any effect on PAPP-A synthesis. In summary, phorbol ester tumor promoters and simian virus 40 transformation regulate IGFBP-4 proteolysis in human fibroblasts through induction of a novel inhibitor of PAPP-A, pro-MBP. These findings expand our understanding of the IGFBP-4 protease system and suggest an additional level of local cell growth control.

We defined the relative contributions of six ligaments in stabilizing the arch of the foot: plantar aponeurosis, long-short plantar ligaments, plantar calcaneonavicular ligament (spring ligament), medial talocalcaneal ligament, talocalcaneal interosseous ligament, and tibionavicular portion of the deltoid ligament. Nineteen fresh-frozen human foot specimens were used. A load of 445 N was applied axially to simulate standing-at-ease posture. Three-dimensional positions of tarsal bones before and after ligament sectioning were determined with the use of a magnetic tracking device. The motions were presented in the form of screw axis displacements, quantitating rotation, and axis of rotation orientation. After sectioning one structure, the arch did not collapse on any specimen and there was no obvious change by visual inspection. There were, however, measurable changes in tarsal bone position. Metatarsal-to-talus total rotation difference was greatest with spring ligament and deltoid ligament sectioning, with an average of 2.1° ± 1.7° and 2.0° ± 0.2° difference, respectively. Calcaneus-to-talus rotation difference was greatest with talocalcaneal interosseous ligament sectioning, with an average of 1.7° ± 1.5°. The spring ligament, deltoid ligament, and talocalcaneal interosseous ligament were most important for arch stability.

We studied six fresh frozen cadaver feet to define the three-dimensional motion of the hallux proximal phalanx in relation to the first metatarsal and to describe the contact features of the first metatarsophalangeal joint. Six tendons to the hallux were loaded to simulate dynamic loading of the hallux. A magnetic tracking system was used to monitor the three-dimensional movement of the proximal phalanx while the toe position was changed from a neutral position to full extension by adjusting the tendon loads. The average surface area was 0.38 ± 0.08 cm 2 in the neutral position; it decreased with toe extension and was the lowest (0.04 ± 0.03 cm 2 ) at the full extension position. Contact distribution of the proximal phalanx did not change substantially throughout the arc of motion. However, for the metatarsal articular surface, the contact distribution shifted dorsally with increasing degrees of extension. These data are consistent with the observation that chondral erosions associated with hallux rigidus and degenerative arthritis initially affect the dorsal articular surface of the metatarsal, and implant arthroplasty often fails from component loading dorsally. The current technique of determining joint contact characteristics is applicable not only for the first metatarsophalangeal joint but also other joints that have not been studied because of shortcomings with more conventional methods.

Primary cilia serve as cellular antenna for various sensory signaling pathways. However, how the sensory receptors are properly targeted to the ciliary surface remains poorly understood. Here, we show that UBC-9, the sole E2 small ubiquitin-like modifier (SUMO)-conjugating enzyme, physically interacts with and SUMOylates the C terminus of small GTPase ARL-13, the worm orthologue of ARL13B that mutated in ciliopathy Joubert syndrome. Mutations that totally abolish the SUMOylation of ARL-13 do not affect its established role in ciliogenesis, but fail to regulate the proper ciliary targeting of various sensory receptors and consequently compromise the corresponding sensory functions. Conversely, constitutively SUMOylated ARL-13 fully rescues all ciliary defects of arl-13–null animals. Furthermore, SUMOylation modification of human ARL13B is required for the ciliary entry of polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease. Our data reveal a novel but conserved role for the SUMOylation modification of ciliary small GTPase ARL13B in specifically regulating the proper ciliary targeting of various sensory receptors.

Abstract Cholangiocarcinomas are rare but aggressive cancers, with an increasing incidence and a dismal 5-year survival rate of less than 5%. Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in ∼70% of gliomas, ∼20% of myeloid leukemias, ∼56% of chondrosarcomas, and ∼10% of melanomas. We discovered IDH1 and IDH2 mutations in 29 of 305 (9%) intrahepatic cholangiocarcinomas. Tumors with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were independently associated with longer time to recurrence of cholangiocarcinoma in multivariate analysis (p = 0.046). We identified 2,267 genes that were significantly hypermethylated in tumors with mutations in IDH1 or IDH2. Hypermethylated CpG sites were significantly enriched in CpG shores and in the 1500 bp upstream of annotated transcription start sites, suggesting a global regulation of transcriptional potential. Gene expression profiling of 7 IDH1/2-mutant and 20 IDH1/2-wildtype tumors revealed increased expression of citric acid cycle and oxidative phosphorylation enzymes, as well as decreased expression of cytoskeleton organization genes. Integrated analyses of DNA methylation and gene expression identified interleukin-6 and several of its downstream targets as candidates for silencing by DNA methylation. Conclusions: Cholangiocarcinomas with mutations in IDH1 or IDH2 represent a molecular subclass with distinct signaling pathway alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1058. doi:1538-7445.AM2012-1058

E-Cadherin–mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P 2 ) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P 2 is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P 2 pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.

Intraflagellar transport (IFT) machinery mediates the bidirectional movement of cargos that are required for the assembly and maintenance of cilia. However, little is known about how IFT is regulated in vivo. In this study, we show that the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor–like protein 13 (ARL-13) encoded by the Caenorhabditis elegans homologue of the human Joubert syndrome causal gene ARL13B, localizes exclusively to the doublet segment of the cilium. arl-13 mutants have shortened cilia with various ultrastructural deformities and a disrupted association between IFT subcomplexes A and B. Intriguingly, depletion of ARL-3, another ciliary small GTPase, partially suppresses ciliogenesis defects in arl-13 mutants by indirectly restoring binding between IFT subcomplexes A and B. Rescue of arl-13 mutants by ARL-3 depletion is mediated by an HDAC6 deacetylase-dependent pathway. Thus, we propose that two conserved small GTPases, ARL-13 and ARL-3, coordinate to regulate IFT and that perturbing this balance results in cilia deformation.