Vitamin D3 inhibits fatty acid synthase expression by stimulating the expression of long-chain fatty-acid-CoA ligase 3 in prostate cancer cells

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.     

The PI3 kinase-Akt pathway mediates Wnt3a-induced proliferation

Wnt3a activates proliferation of fibroblasts cells via activation of both extracellular signal-regulated kinase (ERK) and Wnt/beta-catenin signaling pathways. In this study, we show that the phosphatidyl inositol 3 kinases (PI3K)-Akt pathway is also involved in the Wnt3a-induced proliferation. Akt was activated within 30 min by Wnt3a in NIH3T3 cells. By Wnt3a treatment, activated Akt was transiently accumulated in nucleus although beta-catenin was accumulated in the nucleus of cells in a prolonged manner. The Wnt3a-induced Akt activation was not affected by siRNA-mediated reduction of beta-catenin, indicating that Wnt3a-induced Akt activation may occur independently of beta-catenin. The Wnt3a-induced Akt activation was abolished by pre-treatment with PI3K inhibitor, LY294002 and Wortmanin, but not by MEK inhibitor, U0126, indicating that Wnt3a activates Akt via PI3K. The growth and proliferation induced by Wnt3a were blocked by treatments of the PI3K inhibitors. Furthermore, Wnt3a-induced proliferation was blocked by Akt siRNA. These results reveal that the PI3K-Akt pathway mediates the Wnt3a-induced growth and proliferation of NIH3T3 cells.     

The separation of functionally distinct forms of the third component of human complement (C3).

Complement component C3 prepared by the method of Tack & Prahl [(1976) Biochemistry 15, 4513-4521] was found to contain the following trace contaminants: C3b, haemolytically inactive C3 with intact alpha- and beta-chains (C3u) and degraded C3 (apparent mol.wt. 140000) with an intact beta-chain but with a fragmented alpha-chain. The proportion of C3u in the C3 is increased on standing and by freezing and thawing. These contaminants could be separated from each other and from native C3 by chromatography on sulphated Sepharose. They have been characterized by their susceptibility to C3b inactivator in the presence of beta 1H, their ability to be cleaved by C3 convertase and their ability to form alternative-pathway C3 convertase in solution. Incubation of C3b or C3u with beta 1H and C3b inactivator resulted in cleavage of the C3 species; the alpha'-chain of C3b was cleaved to fragments of apparent mol.wts. 67000 and 43000, the alpha-chain of C3u was cleaved to fragments of apparent mol.wt. 75000 and 43000. Native C3 and degraded C3 were unaffected by incubation with beta 1H and C3b inactivator. C3u, unlike C3, was not cleaved to C3b by the classical- or alternative-pathway C3 convertase in solution. When C3b or C3 was incubated with factors B and D, forming C3 convertase, the initial rate of factor-B cleavage was several order of magnitude lower in the presence of C3 than in the presence of C3b. The slow rate observed for C3 could be decreased by preincubation with beta 1H and C3b inactivator or by rechromatography of the C3. The degraded C3 did not support factor-B cleavage by factor D.     

Interaction of the Rho-ADP-ribosylating C3 exoenzyme with RalA

RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K(d)) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.     

EGF receptor is involved in WNT3a-mediated proliferation and motility of NIH3T3 cells via ERK pathway activation

WNT3a stimulates proliferation of NIH3T3 cells via activation of the extracellular signal-regulated kinase (ERK) pathway. The RAF-1-->MEK-->ERK cascade was immediately increased by WNT3a treatment, however, the upstream event triggering ERK pathway activation by WNT3a is not clear. WNT3a activated RAS and WNT3a-induced ERK activation was blocked by dominant-negative RAS, indicating that WNT3a might act upstream of RAS. WNT3a-induced ERK pathway activations were blocked by AG1478, the epidermal growth factor receptor (EGFR) inhibitor, and EGFR siRNA. The WNT3a-induced ERK pathway activation was not observed in fibroblasts retaining defective EGFR, but the WNT3a effect was restored by EGFR reconstitution. These results indicate involvement of EGFR in the WNT3a-induced ERK pathway activation. WNT3a-induced motility and cytoskeletal rearrangement as well as proliferation of NIH3T3 cells were blocked by AG1478 and EGFR siRNA or abolished in EGFR knock-out fibroblasts, indicating involvement of EGFR in those cellular processes. WNT3a-induced ERK pathway activation was not affected by Dickkoff-1 (DKK-1), although WNT3a-induced activations of the WNT/beta-catenin pathway and proliferation were reduced by DKK-1. EGFR is involved in WNT3a-induced proliferation via both routes dependent on and independent of the WNT/beta-catenin pathway. These results indicate that WNT3a stimulates proliferation and motility of NIH3T3 fibroblasts via EGFR-mediated ERK pathway activation.     

Limited proteolysis of a chemically modified third component of human complement, C3, by cathepsin G of human leukocytes

We have investigated the limited proteolysis of the third component of complement, C3, by a human leukocyte protease, cathepsin G, by using a chemically modified C3, which was prepared by treatment of C3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C3me. Although native C3 was hardly cleaved by cathepsin G, DACM-C3me was cleaved by cathepsin G into three major fragments, which were termed C3c-G (150,000 daltons, 150 kd), C3d-G (25 kd), and C3a-G (10 kd). C3c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C3d-G and C3a-G were single-chain fragments derived from the alpha chain. The N-terminal sequence of C3d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C3d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C3. C3d-G, like C3d-K (a C3d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.     

Induction of fibronectin gene expression by transforming growth factor beta-1 is attenuated in bronchial epithelial cells by ADP-ribosyltransferase inhibitors.

Transforming growth factor-beta (TGF-beta) exerts several effects on cultured airway epithelial cells including inhibition of proliferation and stimulation of fibronectin gene expression. ADP-ribosylation is one potential regulatory mechanism of gene expression by TGF-beta. We tested this possibility by exposing cultured bovine bronchial epithelial cells to the chemical inhibitor of ADP-ribosyl transferase enzymes, 3-aminobenzamide (3-AB) and, for comparison, 3-aminobenzoic acid (3-ABA), which is structurally similar to 3-AB but which does not inhibit ADP-ribosyl transferases. Exponential cell growth rate (1.2 doublings/day) or cellular morphology observed by phase contrast microscopy were not affected by 3 mM 3-AB or 3-ABA. Neither compound antagonized inhibition of cell division or induction of squamous morphology by TGF-beta 1. In contrast, the sixfold stimulation of fibronectin production by exposure of cells to 30 pM TGF-beta 1 for 48 h was reduced by 50% in the presence of 3 mM 3-AB, whereas 3 mM 3-ABA had no effect. The antagonistic effect was augmented by administration of 3-AB 24 h prior to induction by TGF-beta 1. Northern blot hybridization analyses demonstrated that 3-AB, but not 3-ABA, attenuated the induction of fibronectin mRNA by TGF-beta 1 by up to 50%. These observations may implicate a role of cellular ADP-ribosylation in the regulation of some gene expression by TGF-beta.     

Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17alpha,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17alpha-hydroxyvitamin D3 and 17alpha,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 --> 20-hydroxyvitamin D3 --> 20,23-dihydroxyvitamin D3 --> 17alpha,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.     

Adhesive specificity in normal and transformed mouse fibroblasts

Adhesive specificity was studied in normal and transformed $\text{Balb}\text{c}$ mouse fibroblasts by comparing the number of labeled cells collected from a suspension of these cells by aggregates of various cell types. Aggregates of the two malignant cells examined collected either very many cells (aggregates of SV3T3 cells) or very few cells (aggregates of 3T12 cells). In addition, the relative adhesive behavior of these two aggregate types did not vary according to the cell suspension in which they were circulated. These data make it unnecessary to assume that malignancy is always accompanied by a decrease in intercellular adhesion.The adhesive behavior of normal 3T3 cell aggregates, compared to the aggregates composed of either malignant cell type, varied according to the type of cells in the suspension. Aggregates of 3T3 cells collected an appreciable number of SV3T3 cells but few 3T12 cells. Collection of 3T3 cells by 3T3 aggregates was also low if the 3T3 cells of the suspension were harvested from confluent cultures. However, collection of 3T3 cells by 3T3 aggregates increased significantly, as compared to collection by SV3T3 and 3T12 aggregates in the same cell suspension, if the 3T3 suspension was prepared from sparse cultures.Flat-revertants of SV3T3 cells were also studied. These cells behave like nonmalignant 3T3 cells rather than like the SV3T3 cells from which they were derived.We suggest that malignancy may not be caused by decreased intercellular adhesion as compared to normal cells but, perhaps, by decreased intercellular recognition.     

PPARγ activation induces autophagy in breast cancer cells

It has been previously shown that PPARγ ligands induce apoptotic cell death in a variety of cancer cells. Given the evidence that these ligands have a receptor-independent function, we further examined the specific role of PPARγ activation in this biological process. Surprisingly, we failed to demonstrate that MDA-MB-231 breast cancer cells undergo apoptosis when treated with sub-saturation doses of troglitazone and rosiglitazone, which are synthetic PPARγ ligands. Acridine orange (AO) staining showed acidic vesicular formation within ligand-treated cells, indicative of autophagic activity. This was confirmed by autophagosome formation as indicated by redistribution of LC3, an autophagy-specific protein, and the appearance of double-membrane autophagic vacuoles by electron microscopy following exposure to ligand. To determine the mechanism by which PPARγ induces autophagy, we transduced primary mammary epithelial cells with a constitutively active mutant of PPARγ and screened gene expression associated with PPARγ activation by genome-wide array analysis. HIF1α and BNIP3 were among 42 genes up-regulated by active PPARγ. Activation of PPARγ induced HIF1α and BNIP3 protein and mRNA abundance. HIF1α knockdown by shRNA abolished the autophagosome formation induced by PPARγ activation. In summary, our data shows a specific induction of autophagy by PPARγ activation in breast cancer cells providing an understanding of distinct roles of PPARγ in tumorigenesis.     

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro), that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro) protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro) and downstream 3D(pol) sequence, but not 3D(pol) polymerase activity. Cleavage occurs at two non-canonical 3C(pro) recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol) sequence modulates the substrate specificity of the upstream 3C(pro) protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro). HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.     

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

Action of the C3b-inactivator on the cell-bound C3b.

The action of C3bINA and beta 1H on cell-bound C3b is described in this paper. The alpha-polypeptide of C3b that binds covalently to cell surfaces is cleaved by the C3bINA and beta 1H into two fragments: one of 60,000 (C3b alpha-60) and another of 40,000 (C3b alpha-40) daltons. The beta-chain of C3b is unaffected by the C3bINA and beta 1H. The three polypeptides, C3b alpha-60, C3b alpha-40, and C3 beta, are held together as a single unit by disulfide bonds. This unit, referred to as C3b' is covalently bound to cell surfaces via the C3b alpha-60 polypeptide. The conversion of C3b to C3b' by C3bINA and beta 1H abolishes the ability of the C3b-bearing cells to adhere to human erythrocytes as well as the ability to form, on the cell surface, the B, D, and properdin-dependent amplification C3-convertase. However, the agglutinability of the cells with either anti-C3c or anti-C3d is not affected. Treatment of the C3b'-bearing cells with trypsin releases fragments of C3b' into solution, leaving a polypeptide of 32,000 daltons covalently linked to the membrane. Since the trypsinized cells are agglutinable by anti-C3d but not by anti-C3c, the 32,000 dalton polypeptide appears to correspond antigenically to C3d.     

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.     

Tyrosine phosphorylation is an early and requisite signal induced by interferon-γ in HL-60 cells

Interferon-γ (IFNγ) is a potent immunomodulatory cytokine. However, the early mechanisms which mediate the pleiotropic effects of IFNγ on different cells are as yet poorly understood. Therefore, we tested the role of tyrosine phosphorylation in signalling induced by IFNγ. IFNγ was found to induce rapid tyrosine phosphorylation of several proteins in HL-60 cells. This effect was detectable by 2 min, reached a maximum by about 4–16 min and thereafter declined. Tyrosine phosphorylation was dependent on receptor occupation and was maximally stimulated by 10 ng/ml IFNγ. Treatment of HL-60 cells with the tyrosine kinase inhibitors, genistein and herbimycin A, inhibited both IFNγ-stimulated tyrosine phosphorylation and IFNγ-induced Fc receptor expression. Thus, increased tyrosine phosphorylation appears to be an obligatory early and proximal signal mediating at least some of the later cellular responses induced by IFNγ in HL-60 cells.     

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose–phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.     

Glycogen synthase kinase-3 regulates mitochondrial outer membrane permeabilization and apoptosis by destabilization of MCL-1

We investigated the role of glycogen synthase kinase-3 (GSK-3), which is inactivated by AKT, for its role in the regulation of apoptosis. Upon IL-3 withdrawal, protein levels of MCL-1 decreased but were sustained by pharmacological inhibition of GSK-3, which prevented cytochrome c release and apoptosis. MCL-1 was phosphorylated by GSK-3 at a conserved GSK-3 phosphorylation site (S159). S159 phosphorylation of MCL-1 was induced by IL-3 withdrawal or PI3K inhibition and prevented by AKT or inhibition of GSK-3, and it led to increased ubiquitinylation and degradation of MCL-1. A phosphorylation-site mutant (MCL-1(S159A)), expressed in IL-3-dependent cells, showed enhanced stability upon IL-3 withdrawal and conferred increased protection from apoptosis compared to wild-type MCL-1. The results demonstrate that the control of MCL-1 stability by GSK-3 is an important mechanism for the regulation of apoptosis by growth factors, PI3K, and AKT.     

Beta ig-h3 induces keratinocyte differentiation via modulation of involucrin and transglutaminase expression through the integrin alpha3beta1 and the phosphatidylinositol 3-kinase/Akt signaling pathway

Beta ig-h3 is an extracellular matrix protein whose expression is highly induced by transforming growth factor (TGF)-beta1. Whereas beta ig-h3 is known to mediate keratinocyte adhesion and migration, its effects on keratinocyte differentiation remain unclear. In the present study, it was demonstrated that expression of both beta ig-h3 and TGF-beta1 was enhanced during keratinocyte differentiation and that expression of the former was strongly induced by that of the latter. This study also asked whether changes in beta-h3 expression would affect keratinocyte differentiation. Indeed, down-regulation of beta ig-h3 by transfection with antisense beta ig-h3 cDNA constructs effectively inhibited keratinocyte differentiation by decreasing the promoter activities and thus expression of involucrin and transglutaminase. The result was an approximately 2-fold increase in mitotic capacity of the cells. Conversely, overexpression of beta ig-h3, either by transfection with beta ig-h3 expression plasmids or by exposure to recombinant beta ig-h3, enhanced keratinocyte differentiation by inhibiting cell proliferation and concomitantly increasing involucrin and transglutaminase expression. Recombinant beta ig-h3 also promoted keratinocyte adhesion through interaction with integrin alpha3beta1. Changes in beta ig-h3 expression did not affect intracellular calcium levels. Subsequent analysis revealed not only induction of Akt phosphorylation by recombinant beta ig-h3 but also blockage of Akt phosphorylation by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Taken together, these findings indicate that enhanced beta ig-h3, induced by enhanced TGF-beta during keratinocyte differentiation, provoked cell differentiation by enhancing involucrin and transglutaminase expression through the integrin alpha3beta1 and phosphatidylinositol 3-kinase/Akt signaling pathway. Lastly, it was observed that beta ig-h3-mediated keratinocyte differentiation was caused by promotion of cell adhesion and not by calcium regulation.