Identification of sonic hedgehog-responsive genes using cDNA microarray.

Sonic hedgehog (Shh) is a secreted signaling protein that plays important roles in a variety of developmental processes and also in pathogenesis of some human cancers and congenital diseases. Molecules that function downstream of Shh, however, still remain elusive. Here we searched for Shh-responsive genes by using an in-house cDNA microarray. Two genes were newly identified to be Shh responsive in neuroepithelial cell line MNS-70: the metal-binding protein Ceruloplasmin (Cp) and the serine protease inhibitor inter-alpha-trypsine inhibitor heavy chain H3 (ITIH3). In MNS-70 cells, expression of ITIH3 was regulated by Gli zinc-finger transcription factors downstream of Shh, whereas Cp appeared to be regulated by Gli-independent pathways. Cp mRNA was detected in the developing mouse brain, where its expression domain was closely adjacent to that of Shh. These results demonstrate that microarray technology provides a useful tool for studying expression of developmentally regulated genes.

Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1.

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.

Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling.

Gli family zinc finger proteins are mediators of Sonic hedgehog (Shh) signaling in vertebrates. The question remains unanswered, however, as to how these Gli proteins participate in the Shh signaling pathway. In this study, regulatory activities associated with the Gli2 protein were investigated in relation to the Shh signaling. Although Gli2 acts as a weak transcriptional activator, it is in fact a composite of positive and negative regulatory domains. In cultured cells, truncation of the activation domain in the C-terminal half results in a protein with repressor activity, while removal of the repression domain at the N terminus converts Gli2 into a strong activator. In transgenic mouse embryos, N-terminally truncated Gli2, unlike the full length protein, activates a Shh target gene, HNF3beta, in the dorsal neural tube, thus mimicking the effect of Shh signal. This suggests that unmasking of the strong activation potential of Gli2 through modulation of the N-terminal repression domain is one of the key mechanisms of the Shh signaling. A similar regulatory mechanism involving the N-terminal region was also found for Gli3, but not for Gli1. When the Shh signal derived from the notochord is received by the neural plate, the widely expressed Gli2 and Gli3 proteins are presumably converted to their active forms in the ventral cells, leading to activation of transcription of their target genes, including Gli1.

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.