Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.

The Cohesin loading factor NIPBL recruits histone deacetylases to mediate local chromatin modifications.

Cornelia de Lange Syndrome (CdLS) is a rare congenital malformation disorder. About half of the patients with CdLS carry mutations in the NIPBL gene encoding the NIPBL protein, a subunit of the Cohesin loading complex. Recent studies show association of Cohesin with chromatin-remodeling complexes, either by establishing cohesion or by recruiting Cohesin to specific chromosome locations. In yeast two-hybrid assays, we identified an interaction of NIPBL with the histone deacetylases -1 and -3. These interactions were confirmed in mammalian cells by coimmunoprecipitation and a critical region for interaction was defined to a stretch of 163 amino acids of a highly conserved region of NIPBL, which is mutated in patients with CdLS. Utilizing reporter gene assays, we could show that NIPBL fused to the GAL4-DNA-binding domain (GAL4-DBD) represses promoter activity via the recruitment of histone deacetylases. Interestingly, this effect is dramatically reduced by both NIPBL missense mutations identified in CdLS and by chemical inhibition of the histone deacetylases. Our data are the first to indicate a molecular and functional connection of NIPBL with chromatin-remodeling processes via the direct interaction with histone deacetylases.

The origin of the neoplastic stromal cell in giant cell tumor of bone.

Fibroblastlike stromal cells, which are always present as a component of giant cell tumor of bone (GCT), can be observed in both in vivo and cultured cell samples. Although they are assumed to trigger the cancer process in GCT, the histogenesis of GCT stromal cells is poorly understood. It is known that mesenchymal stem cells (MSCs) can develop to osteoblasts. Evidence has been presented that GCT stromal cells can also develop to osteoblasts. A connection between MSCs and GCT stromal cells was sought by using 2 different laboratory approaches. First, immunohistological analyses revealed that some of the same markers, detected by the SH2, SH3, and SH4 antibodies and the CD166 antigen, were found in GCT stromal cells as in the first developmental stages of osteoblast differentiation from the initial MSCs. These immunohistological findings could be confirmed by reverse transcriptase polymerase chain reaction. Second, cellular differentiation by morphology and lineage-specific staining offered evidence that not only osteoblasts, but also chondroblasts and adipocytes, could be cultured from stromal cells. The presented double approach indicates that GCT stromal cells can originate from MSCs.