Foregut malformations (oesophageal atresia, tracheo-oesophageal fistula, lung anomalies and congenital stenosis of the oesophagus and trachea) are relatively common anomalies occurring in 1 in 2,000-5,000 live births, although their aetiology is poorly understood. The secreted glycoprotein Sonic hedgehog (Shh) has been suggested to act as an endodermal signal that controls hindgut patterning and lung growth. In mice, three zinc-finger transcription factors, Gli1, Gli2 and Gli3, have been implicated in the transduction of Shh signal. We report here that mutant mice lacking Gli2 function exhibit foregut defects, including stenosis of the oesophagus and trachea, as well as hypoplasia and lobulation defects of the lung. A reduction of 50% in the gene dosage of Gli3 in a Gli2-/- background resulted in oesophageal atresia with tracheo-oesophageal fistula and a severe lung phenotype. Mutant mice lacking both Gli2 and Gli3 function did not form oesophagus, trachea and lung. These results indicate that Gli2 and Gli3 possess specific and overlapping functions in Shh signalling during foregut development, and suggest that mutations in GLI genes may be involved in human foregut malformations.

The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species [1]; defects in this pathway in humans can cause developmental disorders as well as neoplasia [2]. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood [3] [4]. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins [5] [6], including Costal2, Fused (Fu), and Suppressor of fused (Su(fu)), the last of which has been shown to interact directly with Ci [7]. We have cloned mouse Suppressor of fused (mSu(fu)) and detected its 4.5 kb transcript throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibited transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicated that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity were mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 was localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) resulted in a striking increase in cytoplasmic Gli1 immunostaining. Our findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors.

Macrophage inhibitory cytokine-1 (MIC-1), a divergent member of the TGF-beta superfamily, is involved in the control of multiple cellular processes and mediates cachexia through the inhibition of appetite. Adipose tissue as an endocrine organ secretes proteins (adipokines) that regulate energy homeostasis and other cellular functions. This study investigated whether MIC-1 is expressed in adipose tissue and whether MIC-1 is a secretory product of adipocytes. Mouse and human adipose tissues were collected from different depots. 3T3-L1 preadipocytes and human preadipocytes were induced to differentiate into adipocytes in cell culture. MIC-1 mRNA was detected in the major mouse adipose depots (epididymal, perirenal, sc). In these depots, MIC-1 gene expression was evident in both isolated mature adipocytes and stromal-vascular cells. In 3T3-L1 adipocytes, MIC-1 mRNA was detected before and after differentiation. MIC-1 mRNA and protein secretion were evident in human preadipocytes as well as differentiated adipocytes. MIC-1 production by human adipocytes was stimulated by H(2)O(2) and 15d-prostaglandin J(2). In addition, recombinant MIC-1 increased adiponectin secretion by differentiated human adipocytes. MIC-1 mRNA and protein were also observed in human sc and visceral fat. MIC-1 mRNA levels were positively correlated with adiponectin mRNA. Moreover, MIC-1 mRNA was negatively associated with body mass index and body fat mass in human subjects. We conclude that MIC-1 is expressed in adipose tissue and secreted from adipocytes and is therefore a new adipokine. MIC-1 may have a paracrine role in the modulation of adipose tissue function and body fat mass.

SummaryIntroduction Zinc-a2-glycoprotein (ZAG) is a novel adipokine, which may act locally to influence adipocyte metabolism. This study assessed the effect of increased adiposity on ZAG expression in adipose tissue in human subjects. The study also examined the association between ZAG and adiponectin expression in human adipose tissue, and whether ZAG modulates adiponectin secretion by human adipocytes. Methods Adipose tissue (visceral and subcutaneous) was collected from human subjects with a wide range of BMIs. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were used for in vitro studies. ZAG mRNA levels were quantified by real-time PCR and protein by Western blotting. Results In human subjects, ZAG mRNA level was negatively correlated with BMI (r = )0AE61, P < 0AE001, n = 23, visceral; r = )0AE6, P < 0AE05, n = 14, subcutaneous) and fat mass (r = )0AE62, P < 0AE01, visceral; r = )0AE6, P < 0AE05, subcutaneous). Negative associations were also found between ZAG mRNA and insulin resistance parameters including plasma insulin (r = )0AE65, P < 0AE001, visceral; r = )0AE55, P < 0AE05, subcutaneous) and homeostasis model of insulin resistance (HOMA-IR) (r = )0AE65, P < 0AE001, visceral; r = )0AE52, P = 0AE055, subcutaneous), and C reactive protein (CRP) (r = )0AE46, P < 0AE05, visceral; r = )0AE53, P < 0AE05, subcutaneous). However, ZAG mRNA was positively correlated with adiponectin (r = 0AE5, P < 0AE05, visceral; r = 0AE82, P < 0AE001, subcutaneous) but negatively associated with leptin mRNA (r = )0AE42, P < 0AE05, visceral; r = )0AE54, P < 0AE05, subcutaneous). ZAG secretion by differentiated human adipocytes was abundant. Addition of recombinant ZAG stimulated adiponectin release from human adipocytes. Conclusion ZAG gene expression in adipose tissue is downregulated with increased adiposity and circulating insulin. ZAG mRNA is positively correlated with adiponectin mRNA, and ZAG enhances adiponectin production by human adipocytes. We suggest that ZAG is linked to obesity and obesity-related insulin resistance.

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Suppressor of fused (Su(fu)) is a negative regulator of the Hedgehog signaling pathway that controls the nuclear-cytoplasmic distribution of Gli/Ci transcription factors through direct protein-protein interactions. We show here that Su(fu) is present in a complex with the oncogenic transcriptional activator ␤-catenin and functions as a negative regulator of T-cell factor (Tcf)-dependent transcription. Overexpression of Su(fu) in SW480 (APC mut ) colon cancer cells in which ␤-catenin protein is stabilized leads to a reduction in nuclear ␤-catenin levels and in Tcf-dependent transcription. This effect of Su(fu) overexpression can be blocked by treatment of these cells with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export. Overexpression of Su(fu) suppresses growth of SW480 (APC mut ) tumor cells in nude mice. These observations indicate that Su(fu) negatively regulates ␤-catenin signaling and that CRM-1-mediated nuclear export plays a role in this regulation. Our results also suggest that Su(fu) acts as a tumor suppressor.The oncogenic transcriptional activator ␤-catenin is a major mediator in Wnt signaling (1-4). A large multiprotein complex that includes APC 1 and axin normally facilitates the phosphorylation of ␤-catenin by GSK3␤. Phosphorylated ␤-catenin binds to the F-box protein ␤TrCP and is then modified by ubiquitination and subjected to proteasome-mediated protein degradation. When cells are exposed to the Wnt signal, ␤-catenin phosphorylation and its subsequent ubiquitination are blocked. ␤-Catenin is thus diverted from the proteasome; instead, ␤-catenin accumulates and translocates to the nucleus, where it interacts with members of the Tcf/Lef family of transcription factors and activates transcription of Wnt-responsive genes. In tumors, ␤-catenin degradation is blocked by mutations of APC, axin, or ␤-catenin itself. As a result, stabilized ␤-catenin enters the nucleus and ␤-catenin⅐Tcf complexes activate oncogenic target genes.Nuclear translocation of ␤-catenin is of key importance in its ability to regulate transcription, yet little is known about the factors important in controlling the nuclear versus cytoplasmic distribution of ␤-catenin. ␤-Catenin lacks a nuclear import signal, and it docks to the nuclear membrane by a mechanism that is Ran-independent and does not require importins (5). Nuclear import of ␤-catenin is also independent of its association with the Tcf transcription factors because mutant forms of ␤-catenin that do not bind Tcf proteins can enter the nucleus (6). Microinjection studies show that ␤-catenin rapidly exits the nucleus, suggesting a role for nuclear export in the regulation of the intracellular distribution of ␤-catenin (7).Several studies demonstrate that APC is a nucleo-cytoplasmic protein with export from the nucleus inhibited by LMB, a specific inhibitor of CRM1-mediated nuclear export (8 -10). CRM1, also called exportin-1, is an export karypopherin that binds to a leucine-rich nuclear export signal on its target protein and mediates nuclear-cytoplasm...

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