Asef, a link between the tumor suppressor APC and G-protein signaling.

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. Here the APC gene product is shown to bind through its armadillo repeat domain to a Rac-specific guanine nucleotide exchange factor (GEF), termed Asef. Endogenous APC colocalized with Asef in mouse colon epithelial cells and neuronal cells. Furthermore, APC enhanced the GEF activity of Asef and stimulated Asef-mediated cell flattening, membrane ruffling, and lamellipodia formation in MDCK cells. These results suggest that the APC-Asef complex may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function.

Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3; however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

Cytoplasmic localization of LIM-kinase 1 is directed by a short sequence within the PDZ domain.

LIM-containing protein kinase 1 (LIMK1) is a serine/threonine kinase with a structure composed of two LIM domains, a PDZ domain, and a protein kinase domain. We examined the subcellular localization of LIMK1 and its variously deleted mutants in HeLa cells by transfection with these cDNAs. Immunofluorescence analysis revealed that the full-length LIMK1 and its mutants deleted with LIM domain or protein kinase domain preferentially localized in the cytoplasm, while the mutants deleted with the PDZ domain or a 52 amino acid region (B region) within the PDZ domain localized mainly in the nucleus. When the normally nuclear cyclin A was fused with the PDZ domain or the B region of LIMK1, it was localized in the cytoplasm of transfected cells. The corresponding region of the PDZ domain of postsynaptic density protein (PSD)-95 had no such function. Additionally, the PDZ domain of LIMK1 had no potential to bind to the C-terminal S/TXV peptides, to which the PSD-95 PDZ domain can bind. Taken together these results suggest that the PDZ domain, particularly the B region, of LIMK1 has a specific function to localize the protein in the cytoplasm. When glutathione S-transferase (GST) fused with the PDZ domain of LIMK1 (GST-PDZ) or GST-PDZ deleted with the B region (GST-PDZ delta B) was microinjected into the nucleus of COS cells, GST-PDZ was almost completely excluded from the nucleus within 30 min, whereas GST-PDZ delta B remained in the nucleus. These findings suggest that the B region of LIMK1 probably has nuclear export signal activity.

Serum 1alpha,25-dihydroxyvitamin D3 accumulates into the fracture callus during rat femoral fracture healing.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is thought to be an important systemic factor in the fracture repair process, but the mechanism of action of 1,25(OH)2D3 has not been clearly defined. In this study, the role of 1,25(OH)2D3 in the fracture repair process was analyzed in a rat closed femoral fracture model. The plasma concentration of 1,25(OH)2D3 rapidly decreased on day 3 and continued to decrease to 10 days after fracture. We assessed whether this decrease was based on the accelerated degradation or retardation of the synthesis rate of 1,25(OH)2D3, from 25(OH)D3. After radiolabeled 3H-1,25(OH)2D3 or 3H-25(OH)D3 was injected i.v. into fractured or control (unfractured) rats, the concentrations of 25(OH)D3 and 1,25(OH)2D3 metabolites were measured by HPLC. The plasma concentrations of these radiolabeled metabolites in fractured group were similar to those in control rats early after operation. However, radioactivity in the femurs of fractured rats was higher than that of the control group. Furthermore, the radioactivity was concentrated in the callus of the fractured group analyzed by autoradiography. 1,25(OH)2D3 receptor gene expression was detected early after fracture and, additionally, both in the soft and hard callus on days 7 and 13 after fracture. These results showed that the rapid disappearance of 1,25(OH)2D3 in the early stages after fracture was not due to either increased degradation or decreased synthesis of 1,25(OH)2D3, but rather to increased consumption. Further, these results suggest the possibility that plasma 1,25(OH)2D3 becomes localized in the callus and may regulate cellular events in the process of fracture healing.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Inhibition of activated Ras-induced neuronal differentiation of PC12 cells by the LIM domain of LIM-kinase 1.

LIM-kinase 1 and 2 (LIMK1 and LIMK2) are members of a novel class of protein kinases with structures composed of two LIM motifs at the N-terminus and an unusual protein kinase domain at the C-terminus. The cellular functions of the LIMK family proteins have remained unknown. In the present study, we examined effects of LIMKs on neuronal differentiation of PC12 pheochromocytoma cells. Transient expression analyses revealed that LIMK1, in itself, had no apparent effect on PC12 cells, but the oncogenic Ras-induced differentiation of PC12 cells was notably inhibited by co-expression with LIMK1 or LIMK2. A mutant of LIMK1 lacking a protein kinase domain (delta K) similarly inhibited Ras-induced differentiation of PC12 cells, but a mutant lacking a LIM domain (delta LIM) failed to do so, indicating that a LIM domain but not a protein kinase domain is required for the inhibitory activity. This notion was further supported by the finding that mutation, changing conserved cysteines involved in zinc coordination to glycines in both of two LIM motifs, abolished the inhibitory activity of delta K. Additionally, we also found that the constitutively activated MAP kinase kinase (MAPKK)-induced differentiation of PC12 cells was inhibited by co-expression with delta K. Furthermore, AK did not inhibit the kinase activity of MAP kinase (MAPK) stimulated by MAPKK, when co-expressed in COS7 cells. These findings suggest that LIMK1 inhibits neuronal differentiation of PC12 cells, through its LIM domain and by interfering with events downstream of MAPK activation.

Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain.

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

Protein-protein interaction of zinc finger LIM domains with protein kinase C.

The LIM domain comprising two zinc-finger motifs is found in a variety of proteins and has been proposed to direct protein-protein interactions. During the identification of protein kinase C (PKC)-interacting proteins by a yeast two-hybrid assay, a novel protein containing three LIM domains, designated ENH, was shown to associate with PKC in an isoform-specific manner. Deletion analysis demonstrated that any single LIM domain of ENH associates with the NH2-terminal region of PKC. ENH associated with PKC in COS-7 cells and was phosphorylated by PKC in vitro. Upon treatment of the cells with phorbol ester, ENH in the membrane fraction was translocated to the cytosol fraction in vivo. Other LIM domain-containing proteins, such as Enigma and LIM-kinase 1, also interacted with PKC through their LIM domains. These results suggest that the LIM domain is one of the targets of PKC and that the LIM-PKC interaction may shed light on undefined roles of LIM domain-containing proteins.

Suppression of fibroblast cell growth by overexpression of LIM-kinase 1.

LIM-kinase 1 (LIMK1) is a serine/threonine kinase containing two LIM motifs at the N-terminus. The functional role of LIMK1 has remained unknown. In this study, we examined the role of LIMK1 in cell growth of fibroblasts. Induced expression of LIMK1 in NIH3T3 cells led to growth retardation. Transfection of LIMK1 sense cDNA into NIH3T3 and H-ras-transformed FYJ10 fibroblasts significantly suppressed colony formation of these cells. In contrast, transfection with LIMK1 antisense cDNA strongly stimulated colony formation of the NIH3T3 cells. These findings suggest that LIMK1 functions as a negative regulator of fibroblast cell growth, and may play a role in tumor suppression.

Alendronate modulates osteogenesis of human osteoblastic cells in vitro.

The bisphosphonates, which are carbon-substituted pyrophosphates, have been studied extensively both in vivo and in vitro to elucidate their effects on bone tissues and cells. However, because these agents were shown to have a potent inhibitory effect on bone resorption, the majority of studies have focused on only this aspect of bone metabolism. There appears to be less information regarding the direct effect of bisphosphonates on bone formation, so thus we undertook experiments to investigate the effects of bisphosphonates, especially alendronate, on the mineralization and matrix protein synthesis of human osteoblastic cells in vitro. The data show that the bisphosphonates, alendronate, etidronate and pamidronate, suppressed 1,25-dihydroxycholecalciferol (1,25(OH)2D3)-stimulated mineralization of human osteoblastic cells at high concentrations, while relatively lower concentrations of alendronate and etidronate potentiated mineralization of the cells in the presence of 1,25(OH)2D3. The potentiation of mineralization with alendronate was accompanied by increased synthesis of bone matrix proteins, osteocalcin and collagen, and the mRNA of pro alpha(I) collagen. These findings show that in addition to their well-known effects on bone resorption, bisphosphonates have significant and direct effects on osteogenesis in osteoblasts in vitro. The actual mechanism remains to be further investigated.

Hepatocyte growth factor stimulates growth of hematopoietic progenitor cells.

Hepatocyte growth factor (HGF), a mesenchymal-derived polypeptide, stimulates growth, motility and morphogenesis of various types of cells, most predominantly of epithelial cells. We have now identified an additional biological activity of HGF; this factor probably has a role in early hematopoiesis. We examined the effects of HGF on the growth of various murine hematopoietic progenitor tumor cell lines and found that HGF stimulated DNA synthesis in the myeloid leukemia cell line, NFS-60. The mitogenic effect of HGF on NFS-60 cells was additive with the effect of interleukin 3 (IL-3). On the other hand, HGF had no apparent effect on other myeloid leukemia cell lines examined, such as DA-3 and FDC-P1 cells. Scatchard analysis of specific binding of [125I]HGF revealed expression of a high-affinity HGF receptor on NFS-60 and DA-3 cells, but not on FDC-P1 cells. Expression of c-met mRNA correlated well with the existence of a high-affinity HGF receptor. Since the myeloid leukemia cell lines used are cells in the early stage of myeloid differentiation, HGF may play a role in early hematopoiesis.

Cell density-dependent regulation of hepatocyte growth factor receptor on adult rat hepatocytes in primary culture.

The proliferative potential of hepatocytes in the normal intact liver is highly suppressed, but they proliferate actively after liver injury. In this study, adult rat hepatocytes in primary culture were used to study mechanisms controlling hepatocyte growth in liver regeneration. DNA synthesis in hepatocytes cultured at a low cell density was highly stimulated in response to hepatocyte growth factor (HGF), but this stimulatory effect was not so obvious in hepatocytes cultured at a high cell density. In close parallel to the potency of DNA synthesis, the amounts of 125I-HGF specifically bound to hepatocytes cultured at a low cell density were much greater than in high cell density culture. Scatchard plots revealed that change in the specific binding of 125I-HGF was due to change in the number of high-affinity HGF receptors, but without change in the Kd values. Affinity cross-linking of the HGF receptor with 125I-HGF confirmed the higher expression of HGF receptor on hepatocytes cultured at a lower cell density. Since there was no significant change in the expression of c-met mRNA in hepatocytes cultured at different cell densities, the number of cell surface HGF receptors is probably regulated by post-transcriptional mechanisms. We also found that the rates of HGF-induced down-regulation and recovery of HGF receptors on hepatocytes cultured at a low cell density were much faster than in cases of a high cell density.(ABSTRACT TRUNCATED AT 250 WORDS)

Decrease in the hepatic clearance of hepatocyte growth factor in carbon tetrachloride-intoxicated rats.

To examine whether a decrease in hepatic uptake, clearance or both of hepatocyte growth factor contributes to increased plasma hepatocyte growth factor levels, we kinetically analyzed hepatic hepatocyte growth factor handling using rats with carbon tetrachloride-induced liver injury in both in vivo and perfused liver systems. After the intravenous administration of tracer 125I-hepatocyte growth factor, the time profile of trichloroacetic acid-precipitable 125I-hepatocyte growth factor was analyzed, and tissue clearance and total body plasma clearance were determined. For the tissues examined (liver, kidney, lung, spleen and adrenal), liver and adrenal clearance of 125I-hepatocyte growth factor decreased significantly. It was found that the hepatic clearance explains the bulk of the total body plasma clearance. The hepatic clearance and the total body clearance decreased to minimums (approximately 40% of control) 24 hr after carbon tetrachloride administration and recovered to near-control values over a 6-day period. At 24 hr after carbon tetrachloride administration, a single-pass liver perfusion of 125I-hepatocyte growth factor was performed, and its results were compared with the control results. After a 15-min perfusion of 125I-hepatocyte growth factor, we washed the liver sequentially with heparin and then with acid buffer to separately determine the cell-surface-bound and internalized 125I-hepatocyte growth factor. In carbon tetrachloride-intoxicated rats, both the acid-washable binding and the internalized 125I-hepatocyte growth factor dropped to almost half of the control values, but the decrease in heparin-washable binding was minimal. In contrast, when 125I-hepatocyte growth factor was perfused with excess unlabeled hepatocyte growth factor (135 pmol/L), mostly saturating the cell-surface receptors, the change in cell-surface-bound 125I-hepatocyte growth factor and internalized 125I-hepatocyte growth factor in carbon tetrachloride-intoxicated rats was minimal. This finding, along with our previous finding that the cell-surface hepatocyte growth factor receptors are greatly down-regulated in carbon tetrachloride-intoxicated rats, suggests that the hepatic clearance of hepatocyte growth factor through receptor-mediated endocytosis decreases in carbon tetrachloride-intoxicated rats. The decrease in the hepatic clearance of hepatocyte growth factor could be one of the causes of the elevated hepatocyte growth factor level in the circulating blood in liver diseases.

Importance of the liver in plasma clearance of hepatocyte growth factors in rats.

After intravenous administration of 125I-labeled hepatocyte growth factor (HGF), trichloroacetic acid-precipitable radioactivity in the plasma disappeared rapidly with an early phase half-life of 4 min. The amounts of 125I-HGF distributed to the liver, adrenal, spleen, kidney, and lung tissues were much greater than those that could be accounted for by distribution to the extracellular space alone. The first-pass removal of 125I-HGF by the liver was approximately 26%; the liver accounted for approximately 70% of early-phase removal. The hepatic handling was also analyzed using a single-pass perfused liver system. The steady-state extraction ratio of tracer 125I-HGF was 0.48 but dropped to 0.23 in the presence of excess HGF (135 pM), demonstrating hepatic removal saturation of HGF. In the presence of excess HGF, the heparin-washable 125I-HGF, the heparin-resistant and acid-washable 125I-HGF, and the internalized 125I-HGF dropped to 54, 31, and 32% of the control values. The presence of at least two binding sites for HGF on the liver cell surfaces was made clear: the heparin-washable site and the heparin-resistant and acid-washable binding site, considered to have higher affinity for HGF. The internalization of 125I-HGF was observed to some extent even in the presence of excess HGF and phenylarsine oxide, known to be an inhibitor of polypeptides receptor-mediated endocytosis, suggesting the contribution of a relatively nonspecific internalization mechanism as well as receptor-mediated endocytosis.

Expression of c-met proto-oncogene in COS cells induces the signal transducing high-affinity receptor for hepatocyte growth factor.

By transfection of the expression plasmid containing a human c-met cDNA into COS-7 cells, high-affinity binding sites specific for HGF with a Kd value of 30 pM were newly detected. Furthermore, only in the c-met transfected COS-7 cells, but not in the control COS-7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c-met cDNA resulted in the appearance of high-affinity receptor for HGF and conversion of the normally non-responsive COS-7 cells into the HGF-responsive cells. These results provide evidence for identifying the c-met product as a signal transducing high-affinity receptor for HGF.

Tissue distribution of hepatocyte growth factor receptor and its exclusive down-regulation in a regenerating organ after injury.

Using 125I-labeled hepatocyte growth factor (HGF) as a ligand, we examined the tissue distribution of the HGF receptor in adult rats. Specific binding of 125I-HGF was detected in the plasma membranes of liver, spleen, kidney, lung, adrenal gland, pituitary, and thyroid. Scatchard analysis of HGF binding in liver, spleen, kidney, lung, and adrenal gland revealed the presence of a single class of high affinity receptor with a dissociation constant (Kd) of 20-30 pM. The maximum number of binding sites (Bmax) was determined to be 400-3,000 sites per ng of plasma membrane protein, the highest number being in the liver. Such a wide distribution of a high affinity HGF receptor indicates that HGF may be a multifunctional growth factor, targeting to a variety of organs, and not restricted to liver. After 70% partial hepatectomy, specific binding of 125I-HGF to membranes of the residual liver rapidly decreased, but there was no change in the kidney, lung, and spleen. On the other hand, after unilateral nephrectomy rapid down-regulation of the HGF receptor was clearly evident in the remaining kidney, but not in other organs including the liver. These findings suggest the presence of control mechanisms governing HGF receptor function only in a regenerating organ after injury.