Cleidocranial dysplasia (CCD) is an autosomal dominant disorder characterized by hypoplastic or absent clavicles, large fontanelles, dental anomalies and delayed skeletal development. The phenotype is suggestive of a generalized defect in ossification and is one of the most common skeletal dysplasias not associated with disproportionate stature. To date, no genetic determinants of ossification have been identified. CCD has been mapped to chromosome 6p21, where CBFA1, a gene encoding OSF2/CBFA1, a transcriptional activator of osteoblast differentiation, has been localized. Here, we describe two de novo missense mutations, Met175Arg and Ser191Asn, in the OSF2/CBFA1 gene in two patients with CCD. These two mutations result in substitution of highly conserved amino acids in the DNA-binding domain. DNA-binding studies with the mutant polypeptides show that these amino acid substitutions abolish the DNA-binding ability of OSF2/CBFA1 to its known target sequence. Concurrent studies show that heterozygous nonsense mutations in OSF2/CBFA1 also result in CCD, while mice homozygous for the osf2/cbfa1 mull allele exhibit a more severe lethal phenotype. Thus, these results together suggest that CCD is produced by haploinsufficiency of OSF2/CBFA1 and provide direct genetic evidence that the phenotype is secondary to an alteration of osteoblast differentiation.

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G-protein-coupled receptor (GPCR) kinases, GRKs, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5-IB␣ interaction on NFB signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of IB␣, leading to the inhibition of NFB transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and IB␣, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the N-terminal domain of IB␣ as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NFB, we evaluated the effects of GRK5-RH on NFB-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NFB transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NFB activity.angiogenesis ͉ gene transcription ͉ inflammation ͉ signal transduction G -protein-coupled receptor (GPCR) kinases, GRKs, constitute a large family of serine/threonine protein kinases that regulate GPCR signaling (1-3), consisting of 7 isoforms that share structural and functional similarities (4). A central catalytic domain is flanked by an N-terminal domain that includes a region of homology to regulators of G-protein signaling (RH) and a C-terminal domain of variable length (5, 6). The catalytic domain of GRKs is relatively well-conserved among the members of different subfamilies (Ϸ45% sequence identity), whereas N-terminal RH domains display weak homology (Ϸ27%) and C termini have little or no sequence homology. GRKs have different tissue distribution, subcellular localization, and kinase activity regulation (7,8). GRKs mostly localize at the plasma membrane (2), but recently Johnson et al. (7) demonstrated that GRK4-6 (but not other GRKs) can shuttle between cytosol and nucleus through functional nuclear localization sequence (NLS) and nuclear exporting sequence (NES), thus suggesting a nuclear effect for the GRK4-6 subfamily.NFB is an ubiquitously expressed and highly regulated dimeric transcription factor (3) regulating the expression of genes responsible for innate and adaptive immunity, tissue regeneration, stress responses, apoptosis, cell proliferation, and differentiation (9-14). Within the canonical view of the regulation of the NFB activity, the inactive NFB/IB␣ complex localizes in the cytosol until an extracellular stimulus, ...

Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription.