Transforming growth factor β (TGFβ) is important in inflammation, angiogenesis, reepithelialization and connective tissue regeneration during wound healing. We analyzed components of TGFβ signaling pathway in biopsies from 10 patients with nonhealing venous ulcers (VUs). Using comparative genomics of transcriptional profiles of VUs and TGFβ-treated keratinocytes, we found deregulation of TGFβ target genes in VUs. Using quantitative polymerase chain reaction (qPCR) and immunohistochemical analysis, we found suppression of TGFβRI, TGFβRII and TGFβRIII, and complete absence of phosphorylated Smad2 (pSmad2) in VU epidermis. In contrast, pSmad2 was induced in the cells of the migrating epithelial tongue of acute wounds. TGFβ-inducible transcription factors (GADD45β, ATF3 and ZFP36L1) were suppressed in VUs. Likewise, genes suppressed by TGFβ (FABP5, CSTA and S100A8) were induced in nonhealing VUs. An inhibitor of Smad signaling, Smad7 was also downregulated in VUs. We conclude that TGFβ signaling is functionally blocked in VUs by downregulation of TGFβ receptors and attenuation of Smad signaling resulting in deregulation of TGFβ target genes and consequent hyperproliferation. These data suggest that application of exogenous TGFβ may not be a beneficial treatment for VUs.

An unconventional TGFβ superfamily pathway plays a crucial role in the decision between dauer diapause and reproductive growth. We have studied the daf-5 gene, which, along with the daf-3 Smad gene, is antagonized by upstream receptors and receptor-regulated Smads. We show that DAF-5 is a novel member of the Sno/Ski superfamily that binds to DAF-3 Smad, suggesting that DAF-5, like Sno/Ski, is a regulator of transcription in a TGFβ superfamily signaling pathway. However, we present evidence that DAF-5 is an unconventional Sno/Ski protein, because DAF-5 acts as a co-factor, rather than an antagonist, of a Smad protein. We show that expressing DAF-5 in the nervous system rescues a daf-5 mutant, whereas muscle or hypodermal expression does not.Previous work suggested that DAF-5 and DAF-3 function in pharyngeal muscle to regulate gene expression, but our analysis of regulation of a pharynx specific promoter suggests otherwise. We present a model in which DAF-5 and DAF-3 control the production or release of a hormone from the nervous system by either regulating the expression of biosynthetic genes or by altering the connectivity or the differentiated state of neurons.

Protein geranylgeranyltransferase-I (PGGT-I) and protein farnesyltransferase (PFT) attach geranylgeranyl and farnesyl groups, respectively, to the C termini of eukaryotic cell proteins. In vitro, PGGT-I and PFT can transfer both geranylgeranyl and farnesyl groups from geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP) to their protein or peptide prenyl acceptor substrates. In the present study it is shown that PGGT-I binds GGPP 330-fold tighter than FPP and that PFT binds FPP 15-fold tighter than GGPP. Therefore, in vivo, where both GGPP and FPP compete for the binding to prenyltransferases, PGGT-I and PFT will likely be bound predominantly to GGPP and FPP, respectively. Previous studies have shown that K-Ras4B and the Ras-related GTPase TC21 are substrates for both PGGT-I and PFT in vitro. It is shown that TC21 can compete with the C-terminal peptide of the ␥ subunit of heterotrimeric G proteins and with the C-terminal peptide of lamin B for geranylgeranylation by PGGT-I and for farnesylation by PFT, respectively. K-Ras4B competes in both cases but is almost exclusively farnesylated by PFT in the presence of the lamin B peptide competitor. Rapid and single turnover kinetic studies indicate that the rate constant for the PGGT-I-catalyzed geranylgeranyl transfer step of the reaction cycle is 14-fold larger than the steady-state turnover number, which indicates that the rate of the overall reaction is limited by a step subsequent to prenyl transfer such as release of products from the enzyme. PGGT-I-catalyzed farnesylation is 37-fold slower than geranylgeranylation and is limited by the farnesyl transfer step. These results together with earlier studies provide a paradigm for the substrate specificity of PGGT-I and PFT and provide information that is critical for the design of prenyltransferase inhibitors as anti-cancer agents.Modification of the C termini of specific eukaryotic proteins by attachment of either 15-carbon farnesyl or 20-carbon geranylgeranyl groups is required for their proper membrane targeting and functional activation (1-9). Two closely related enzymes protein farnesyltransferase (PFT) 1 and PGGT-I transfer prenyl groups from prenyl pyrophosphates to proteins that contain a C-terminal CaaX motif (C is cysteine, a is usually an aliphatic amino acid, and X is a variety of amino acids) (10 -13). The X residue of this motif plays a major role in recognition by these two enzymes (14). PFT preferentially transfers a farnesyl group to the cysteine residue of the CaaX motif when X is serine, methionine, glutamine, or cysteine, and possibly other residues. PGGT-I preferentially geranylgeranylates proteins having a C-terminal leucine or phenylalanine. PFT and PGGT-I consist of a common ␣ subunit and distinct ␤ subunits (15-18). A third enzyme, protein geranylgeranyltransferase-II, also known as Rab geranylgeranyltransferase, transfers the 20-carbon prenyl group to both cysteines of Rab proteins that have C-terminal sequences CXC, CC, or possibly CCXX (19,20). Although the C-terminal CaaX...

Background: When resources are scant, C. elegans larvae arrest as long-lived dauers under the control of insulin/IGF-and TGFβ-related signaling pathways. However, critical questions remain regarding the regulation of this developmental event. How do three dozen insulin-like proteins regulate one tyrosine kinase receptor to control complex events in dauer, metabolism and aging? How are signals from the TGFβ and insulin/IGF pathways integrated? What gene expression programs do these pathways regulate, and how do they control complex downstream events?

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