Activin induces long-lasting N-methyl-D-aspartate receptor activation via scaffolding PDZ protein activin receptor interacting protein 1.

Calcium entry into the postsynaptic neuron through N-methyl-D-aspartate-type glutamate receptors (NMDARs) triggers the induction of long-term potentiation (LTP), which is considered to contribute to synaptic plasticity and plays a critical role in behavioral learning. We report here that activin, a member of the transforming growth factor-beta (TGF-beta) superfamily, promotes phosphorylation of NMDARs and increases the Ca2+ influx through these receptors in primary cultured rat hippocampal neurons. This signal transduction occurs in a functional complex of activin receptors, NMDARs, and Src family tyrosine kinases, including Fyn, formed on a multimer of postsynaptic scaffolding postsynaptic density protein 95/Dlg/ZO-1 (PDZ), activin receptor interacting protein 1 (ARIP1). Activin-induced NMDAR activation persists for more than 24 h, which is complimentary to the activation time of NMDARs by brain-derived neurotrophic factor (BDNF). Our results suggest that activin is a unique and powerful potentiator for NMDAR-dependent signaling, which could be involved in the regulatory mechanisms of synaptic plasticity.

Characterization of isoforms of activin receptor-interacting protein 2 that augment activin signaling.

Activin type II receptors (ActRIIs) including ActRIIA and ActRIIB are serine/threonine kinase receptors that form complexes with type I receptors to transmit intracellular signaling of activins, nodal, myostatin and a subset of bone morphogenetic proteins. ActRIIs are unique among serine/threonine kinase receptors in that they associate with proteins having PSD-95, Discs large and ZO-1 (PDZ) domains. In our previous studies, we reported specific interactions of ActRIIs with two independent PDZ proteins named activin receptor-interacting proteins 1 and 2 (ARIP1 and ARIP2). Overexpression of both ARIP1 and ARIP2 reduce activin-induced transcription. Here, we report the isolation of two isoforms of ARIP2 named ARIP2b and 2c. ARIP2, ARIP2b and ARIP2c recognize COOH-terminal residues of ActRIIA that match a PDZ-binding consensus motif. ARIP2 and its isoforms have one PDZ domain in the NH2-terminal region, and interact with ActRIIA. Although PDZ domains containing GLGF motifs of ARIP2b and 2c are identical to that of ARIP2, their COOH-terminal sequences differ from that of ARIP2. Interestingly, unlike ARIP2, overexpression of ARIP2b or 2c did not affect ActRIIA internalization. ARIP2b/2c inhibit inhibitory actions of ARIP2 on activin signaling. ARIP2 is widely distributed in mouse tissues. ARIP2b/2c is expressed in more restricted tissues such as heart, brain, kidneys and liver. Our results indicate that although both ARIP2 and ARIP2b/2c interact with activin receptors, they regulate ActRIIA function in a different manner.

FKBP12 functions as an adaptor of the Smad7-Smurf1 complex on activin type I receptor.

The cytoplasmic immunophilin FKBP12, a 12 kDa FK506-binding protein, has been shown to act as an inhibitor for transforming growth factor-beta (TGF-beta) signaling. FKBP12 binds to the glycine- and serine-rich motif (GS motif) of the TGF-beta type I receptor, and functions as a secure switch to prevent the leaky signal. Upon stimulation with ligand, FKBP12 is released from the receptor to fully propagate the signal. We found that activin, a member of TGF-beta superfamily, also induced the dissociation of FKBP12 from the activin type I receptor (ALK4). However, we observed that the released FKBP12 associates again with the receptor a few hours later. FKBP12 also interacted with another inhibitory molecule of activin signal, Smad7, in an activin-dependent manner, and formed a complex with Smad7 on the type I receptor. FK506, a chemical ligand for FKBP12, which dissociates FKBP12 from the receptor, decreased the interaction between Smad7 and Smad ubiquitin regulatory factor 1 (Smurf1). FK506 also inhibited the ubiquitination of the type I receptor by Smurf1. These findings indicate a new inhibitory function of FKBP12 as an adaptor molecule for the Smad7-Smurf1 complex to regulate the duration of the activin signal.

Synergistic activity of activin A and basic fibroblast growth factor on tyrosine hydroxylase expression through Smad3 and ERK1/ERK2 MAPK signaling pathways.

Activin has previously been shown to act as a nerve cell survival factor and to have neurotrophic effects on neurons. However, the role of activin in regulating neurotransmitter expression in the central nervous system and the exact mechanisms involved in this process are poorly understood. In the present study, we report that activin A and basic fibroblast growth factor (bFGF) synergistically increased the protein level of tyrosine hydroxylase (TH), and also greatly increased the TH mRNA level, in both mouse E14 striatal primary cell cultures and the hippocampal neuronal cell line HT22. Activin A and bFGF cooperatively stimulated nuclear translocation of Smad3 and specifically activated ERK1/2, but not p38 or JNK. Interestingly, a specific inhibitor for MEK, U0126, efficiently blocked the induction of TH promoter activity by activin A and bFGF, indicating that activin A collaborated with bFGF signaling to induce the TH gene through selective activation of ERK-type MAP kinase in mouse striatal and HT22 cells. These data suggest that activin A may act in concert with bFGF for the development of TH-positive neurons.

Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner.

Smad proteins are cytoplasmic signaling effectors of transforming growth factor-beta (TGF-beta) family cytokines and regulate gene transcription in the nucleus. Receptor-activated Smads (R-Smads) become phosphorylated by the TGF-beta type I receptor. Rapid and precise transport of R-Smads to the nucleus is of crucial importance for signal transduction. By focusing on the R-Smad Smad3 we demonstrate that 1) only activated Smad3 efficiently enters the nucleus of permeabilized cells in an energy- and cytosol-dependent manner. 2) Smad3, via its N-terminal domain, interacts specifically with importin-beta1 and only after activation by receptor. In contrast, the unique insert of exon3 in the N-terminal domain of Smad2 prevents its association with importin-beta1. 3) Nuclear import of Smad3 in vivo requires the action of the Ran GTPase, which mediates release of Smad3 from the complex with importin-beta1. 4) Importin-beta1, Ran, and p10/NTF2 are sufficient to mediate import of activated Smad3. The data describe a pathway whereby Smad3 phosphorylation by the TGF-beta receptor leads to enhanced interaction with importin-beta1 and Ran-dependent import and release into the nucleus. The import mechanism of Smad3 shows distinct features from that of the related Smad2 and the structural basis for this difference maps to the divergent sequences of their N-terminal domains.

Role of Smad proteins and transcription factor Sp1 in p21(Waf1/Cip1) regulation by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21(WAF1/Cip1) (p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-beta, Smad3 and Smad4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that Smad3 and Smad4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaT p21 gene by TGF-beta1 is further enhanced after overexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad3 and Smad4 and the inhibitory Smad7 all inhibit p21 induction by TGF-beta1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas Smad proteins do not. In support of this finding, we show that DNA-binding domain mutants of Smad3 and Smad4 are capable of transactivating the p21 promoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4 and Sp1 in SL-2 cells or co-incubation of phosphorylated Smad3, Smad4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with Smad2, Smad3, and weakly with Smad4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with Smad proteins. In conclusion, Smad proteins play important roles in regulation of the p21 gene by TGF-beta, and the functional cooperation of Smad proteins with Sp1 involves the physical interaction of these two types of transcription factors.

Expression of a TGF-beta1 inducible gene, TSC-36, causes growth inhibition in human lung cancer cell lines.

TSC-36 (TGF-beta1-stimulated clone 36) is a TGF-beta1 inducible gene whose product is an extracellular glycoprotein that contains a single follistatin module. TSC-36 is highly expressed in the lung, but its physiological function is unknown. In an attempt to elucidate it, we investigated the effect of TSC-36 on proliferation of human lung cancer cell lines. We found a correlation between expression of TSC-36 and cell growth: TSC-36 mRNA was not detected in cells derived from small cell lung cancer (SCLC) cells, a highly aggressive neoplasm, but was detected in some non-small cell lung cancer (NSCLC) cells, a moderately aggressive neoplasm. This suggested an antiproliferative function for TSC-36. To address this question, NSCLC PC-14 cells, which express very low level of TSC-36 protein, were transfected with TSC-36 cDNA and the proliferative capacity of stable transfectants was determined by measuring the doubling time, colony forming activity in soft agar and the level of incorporation of (3)H-thymidine into DNA. Under normal culture conditions, the transfected cells showed a longer doubling time, lower plating efficiency and lower rate of DNA synthesis than the parental cells and the control neo transfectant cells. These findings suggested that expression of TSC-36 caused growth inhibition in human lung cancer cells.

TGF-(beta) type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells.

The capacities of different transforming growth factor-(beta) (TGF-(beta)) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-(beta)1, but not activin A or osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7), was able to induce morphological transformation of NMuMG cells as shown by reorganisation of the actin cytoskeleton and relocalisation/downregulation of E-cadherin and (beta)-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-(beta)1 bound to TGF-(beta) type I receptor (T(beta)R-I)/ALK-5 and T(beta)R-II, but not to activin type I receptor (ActR-I)/ALK-2. Activin A bound to ActR-IB/ALK-4 and ActR-II, and BMP-7 bound to ActR-I/ALK-2, BMP type I receptor (BMPR-I)/ALK-3, ActR-II and BMPR-II. TGF-(beta)1 and BMP-7 activated the Smad-binding element (SBE)(4) promoter with equal potency, whereas activin A had no effect. Transfection of constitutively active (CA)-ALK-4 activated the 3TP promoter to the same extent as TGF-(beta)1 and CA-ALK-5 indicating that activin signalling downstream of type I receptors was functional in NMuMG cells. In agreement with this, activin A induced low levels of plasminogen activator inhibitor I expression compared to the high induction by TGF-(beta)1. In contrast to activin A and BMP-7, TGF-(beta)1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-(beta)1 induced the nuclear accumulation of Smad2 and/or Smad3. In addition, NMuMG cells transiently infected with adenoviral vectors expressing high level CA-ALK-5 exhibited full transdifferentiation. On the other hand, infections with low level CA-ALK-5, which alone did not result in transdifferentiation, together with Smad2 and Smad4, or with Smad3 and Smad4 led to transdifferentiation. In conclusion, TGF-(beta)1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-(beta) type I receptor (ALK-5) and its effector Smad proteins mediate the epithelial to mesenchymal transition. Activin A does not induce mesenchymal transformation, presumably because the number of activin receptors is limited, while BMP-7-initiated signalling cannot mediate transdifferentiation.

A partial deficiency of dehydrodolichol reduction is a cause of carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is a congenital disorder that involves the underglycosylation of N-glycosylated glycoproteins (Yamashita, K., Ideo, H., Ohkura, T., Fukushima, K., Yuasa, I., Ohno, K., and Takeshita, K. (1993) J. Biol. Chem. 268, 5783-5789). In an effort to further elucidate the biochemical basis of CDG syndrome type I in our patients, we investigated the defect in the multi-step pathway for biosynthesis of lipid-linked oligosaccharides (LLO) by the metabolic labeling method using [3H]glucosamine, [3H]mannose, and [3H]mevalonate. The LLO levels in synchronized cultures of fibroblasts from these patients were severalfold lower than those in control fibroblasts in the S phase, and the oligosaccharides released from LLO showed the same structural composition, Glc1 approximately 3.Man9.GlcNAc.GlcNAc, in the case of both the patients and controls. The amount of [3H]mannose incorporated into mannose 6-phosphate, mannose 1-phosphate, and GDP-mannose was greater in fibroblasts from these patients than in the control fibroblasts in the G1 period, although the ratios of these acidic mannose derivatives as indicated by the relative levels of radioactivity were the same for the two types of fibroblasts. Furthermore, upon metabolic labeling with [3H]mevalonate, the level of [3H]dehydrodolichol in fibroblasts from these patients increased in the S phase, and the levels of [3H]dolichol and [3H]dolichol-PP oligosaccharides concomitantly decreased, although the chain length distribution of the respective dolichols and dehydrodolichols was the same in the two types of fibroblasts. These results indicate that the conversion of dehydrodolichol to dolichol is partially defective in our patients and that the resulting loss of dolichol leads directly to underglycosylation.

Proteolytic release of dehydrodolichyl diphosphate synthase from pig testis microsomes.

Pig testicular dehydrodolichyl diphosphate synthase was released in a soluble form out of microsomes by controlled proteolysis with trypsin or papain. Approximately 25% of the microsomal enzyme activity was recovered in the 115,000 x g supernatant fraction when the microsomes were treated with trypsin at 4 degrees C for 1 h. Similar proteolytic release of microsomal enzyme was also observed with the treatment with papain. The K(m), optimal pH, Mg2+ dependency, and ion strength dependency of the enzyme released by trypsin were similar to those of the microsomal enzyme. The microsomal enzyme was active even in the absence of detergents, while the released enzyme required detergents for activity. Gel filtration of the released enzyme gave a peak of dehydrodolichyl diphosphate synthase activity, which appeared between 150-kDa and 50-kDa molecular mass markers.

Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.

When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.

Formation of dolichol from dehydrodolichol is catalyzed by NADPH-dependent reductase localized in microsomes of rat liver.

The alpha-saturation reaction involved in the biosynthesis of dolichol has been investigated with rat liver preparations. Under improved in vitro conditions with 10,000 x g supernatant of rat liver homogenates in the presence of NADPH at pH 8.0, dolichol was synthesized from isopentenyl diphosphate and Z,E,E-geranylgeranyl diphosphate. Neither dolichyl diphosphate nor dolichyl phosphate was detected. The chain length distribution of the dolicohol was the same as that of dehydrodolichyl products. In an assay system containing dehydrodolichol, dehydrodolichyl phosphate, or dehydrodolichyl diphosphate as a substrate, dehydrodolichol was predominantly converted into dolichol. The enzyme that catalyzes the conversion of dehydrodolichol to dolichol was localized in microsomes. The reductase activity was stimulated 9-fold by the addition of a 100,000 x g soluble fraction. The reductase had an opimal pH at 8.0. These results indicate that dolichol is formed from dehydrodolichol in rat liver microsomes. The formation of dolichol from dehydrodolichol was also catalyzed by 10,000 x g supernatant of rat or pig testis homogenates.

Separation of dolichol from dehydrodolichol by a simple two-plate thin-layer chromatography.

A novel thin-layer chromatographic procedure was devised to separate dolichol and dehydrodolichol from each other with the concomitant separation of each family with respect to the carbon chain length. This method involves development of the polyprenols successively on two different plates, a silica gel plate and a reversed-phase plate.

Variable product specificity of microsomal dehydrodolichyl diphosphate synthase from rat liver.

Several detergents activated microsomal dehydrodolichyl diphosphate synthase of rat liver, but the chain length of products shifted downward from C90 and C95 with increasing concentration of the detergents. Maximum activation was observed at the concentration of 2% Triton X-100, 30 mM octyl glucoside, 30 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 10 mM deoxycholate with the product chain length being C80-C85, C65-C75, C70-C75, and C55-C65, respectively. The activity of Triton X-100 solubilized enzyme was decreased by asolectin, phosphatidylethanolamine, and phosphatidylcholine. The chain lengths of products formed in the presence of these phospholipids were C85 and C90. In the presence of both phosphatidylcholine and Mg2+ the solubilized enzyme was able to produce C90 and C95 dehydrodolichyl diphosphates like native microsomal enzyme. Microsomal enzyme preparations from rat liver, brain, and testis catalyzed the formation of dehydrodolichyl diphosphates with the same chain lengths as those of the natural dolichols occurring in individual tissues. The chain length distribution of dehydrodolichyl products by (rat liver) microsomes also depended on the concentration of substrates. Not only did increasing the concentration of isopentenyl diphosphate lead to longer chain product, but decreasing that of farnesyl diphosphate increased product chain length.