Transforming growth factor-␤1 (TGF-␤1) is the most abundant TGF-␤ isoform detected in bone and is an important functional modulator of osteoclasts. TGF-␤1 can induce osteoclast apoptosis; however, the apoptotic pathways involved in this process are not known. We show here that human osteoclasts express both type-I and type-II TGF-␤ receptors. In the absence of survival factors, TGF-␤1 (1 ng/ml) induced osteoclast apoptosis. The expression of activated caspase-9, but not that of caspase-8, was increased by TGF-␤1 stimulation, and the rate of TGF-␤1-induced apoptosis was significantly lower in the presence of a caspase-9 inhibitor. To study further the mechanisms involved in TGF-␤1-induced osteoclast apoptosis, we investigated TGF-␤1 signaling, which primarily involves the Smad pathway, but also other pathways that may interfere with intracellular modulators of apoptosis, such as mitogen-activated protein (MAP) kinases and Bcl2 family members. We show here that early events consisted of a trend toward increased expression of extracellular signal-regulated kinase (ERK), and then TGF-␤1 significantly induced the activation of p38 and Smad2 in a time-dependent manner. These signaling cascades may activate the intrinsic apoptosis pathway, which involves Bim, the expression of which was increased in the presence of TGF-␤1. Furthermore, the rate of TGF-␤1-induced osteoclast apoptosis was lower when Bim expression was suppressed, and inhibiting the Smad pathway abolished Bim up-regulation following TGF-␤ stimulation. This could correspond to a regulatory mechanism involved in the inhibition of osteoclast activity by TGF-␤1.

In their letter, Labrinidis and colleagues raise the important issue of what (if any) might be the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in osteoclastogenesis. In previous studies, we 1,2 and 2 other independent groups of investigators 3-5 have shown that histidine-tagged (His-tag) recombinant TRAIL negatively regulates osteoclastogenesis by inhibiting preosteoclast differentiation and by inducing apoptosis of mature osteoclasts. On the other hand, Labrinidis et al were unable to confirm these previous findings when exposing osteoclastic cultures to the version of Apo2L/TRAIL that is currently being used in phase 1b clinical trials. Although Labrinidis et al emphasize the differences between the recombinant TRAIL preparations used in their and previous studies, 1-5 the possibility that the antiosteoclastic activity of TRAIL merely reflects an aspecific toxic effect of recombinant His-tag TRAIL is ruled out by 2 major considerations: (1) different groups of investigators have clearly documented the ability of recombinant His-TRAIL to induce in vitro prosurvival and even proliferative responses in a cell-type specific manner 6 ; and (2) Roux's group has recently demonstrated that native TRAIL, produced and released in vitro by end-stage osteoclasts, promotes osteoclastic apoptosis through autocrine/paracrine mechanism. 4 Thus, besides blocking receptor-activator of NF-B ligand (RANKL)-mediated osteoclastogenesis, osteoprotegerin (OPG) seems also able to protect mature osteoclasts from apoptosis mediated by native TRAIL endogenously produced by osteoclasts. 4 These findings corroborate the hypothesis that the relative concentrations of RANKL, OPG, and TRAIL at the local bone marrow level are critical for determining the fate of osteoclasts. 6,7 The net effect of TRAIL on osteoclastic differentiation and survival likely depends on the network of prosurvival and proapoptotic signals operating at a given time in the bone marrow microenvironment. In this respect, it should be considered that the antiosteoclastic activity of TRAIL reported by our and other groups 1-5 was observed in culture conditions in which purified populations of preoste-oclasts were induced to differentiate along the osteoclastic lineage by adding recombinant macrophage-colony stimulating factor (M-CSF) plus RANKL to the culture medium. On the other hand, Labrinidis et al have cultured peripheral blood mononuclear cells (used as a source of preosteoclasts) in the presence also of vitamin D3 and dexamethasone, which are known to potently promote osteclastic survival and differentiation. 8 Thus, in our view, the novel contribution of the findings of Labrinidis et al with respect to previous data 1-5 relies on the demonstration that the presence in culture of vitamin D3 and dexamethasone abrogates the antidiffer-entiative and proapoptotic activities of TRAIL. However, this does not exclude a role of TRAIL in osteoclastogenesis, as suspected by Labrinidis et al, but rather suggests a level of molecular control on the a...

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.

is essential for development of several organs and tissues. In the respiratory system, loss of function causes neonatal death due to respiratory distress. Expression of HOXA5 protein in mesenchyme of the respiratory tract and in phrenic motor neurons of the central nervous system led us to address the individual contribution of these expression domains using a conditional gene targeting approach. does not play a cell-autonomous role in lung epithelium, consistent with lack of HOXA5 expression in this cell layer. In contrast, ablation of in mesenchyme perturbed trachea development, lung epithelial cell differentiation and lung growth. Further, deletion of in motor neurons resulted in abnormal diaphragm innervation and musculature, and lung hypoplasia. It also reproduced the neonatal lethality observed in null mutants, indicating that the defective diaphragm is the main cause of impaired survival at birth. Thus, possesses tissue-specific functions that differentially contribute to the morphogenesis of the respiratory tract.