Effect of glycosaminoglycan structure on all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

Glycosaminoglycan polysaccharides are components of animal extracellular matrices and regulate cell functions based on their various sulfation and epimerization pattern structures. The present study aimed to find glycosaminoglycan structures to promote neural differentiation. We investigated the effect of exogenous glycosaminoglycans with well-defined structures on the all-trans-retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells, which is an ideal model culture system for studying neural differentiation. We found that chondroitin sulfate E and heparin, but not any other glycosaminoglycans, upregulated the expressions of neural specific markers but not a grail specific marker. Chondroitin sulfate E was suggested to function during spheroid formation, however, equimolar concentration of its oligosaccharide did not show promotive effect on the neural differentiation. Another finding was that hyaluronan oligosaccharide mixture markedly downregulated the expressions of a myelin specific marker. These findings suggested that the specific sulfation pattern and/or chain length of exogenous added glycosaminoglycan is important to regulate neural differentiation and myelination.

The expression of Fn14 via mechanical stress-activated JNK contributes to apoptosis induction in osteoblasts.

Bone mass is maintained by the balance between the activities of bone-forming osteoblasts and bone-resorbing osteoclasts. It is well known that adequate mechanical stress is essential for the maintenance of bone mass, whereas excess mechanical stress induces bone resorption. However, it has not been clarified how osteoblasts respond to different magnitudes of mechanical stress. Here we report that large-magnitude (12%) cyclic stretch induced Ca(2+) influx, which activated reactive oxygen species generation in MC3T3-E1 osteoblasts. Reactive oxygen species then activated the ASK1-JNK/p38 pathways. The activated JNK led to transiently enhanced expression of FGF-inducible 14 (Fn14, a member of the TNF receptor superfamily) gene. Cells with enhanced expression of Fn14 subsequently acquired sensitivity to the ligand of Fn14, TNF-related weak inducer of apoptosis, and underwent apoptosis. On the other hand, the ASK1-p38 pathway induced expression of the monocyte chemoattractant protein 3 (MCP-3) gene, which promoted chemotaxis of preosteoclasts. In contrast, the ERK pathway was activated by small-magnitude stretching (1%) and induced expression of two osteogenic genes, collagen Ia (Col1a) and osteopontin (OPN). Moreover, activated JNK suppressed Col1a and OPN induction in large-magnitude mechanical stretch-loaded cells. The enhanced expression of Fn14 and MCP-3 by 12% stretch and the enhanced expression of Col1a and OPN by 1% stretch were also observed in mouse primary osteoblasts. These results suggest that differences in the response of osteoblasts to varying magnitudes of mechanical stress play a key role in switching the mode of bone metabolism between formation and resorption.

N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells.

PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α subunit of AMPK (AMPKα) in cells. N-Myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity towards AMPKα. In in vitro experiments, the G2A mutants exhibited reduced activities towards AMPKα, but much higher specific activity against an artificial substrate, PNPP (p-nitrophenyl phosphate), compared with the wild-type counterparts. Taken together, the results of the present study suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.

TGF-ß-activated kinase 1 mediates mechanical stress-induced IL-6 expression in osteoblasts.

Mechanical stress plays a key role in bone remodeling. Previous studies showed that loading of mechanical stretch induces a rapid Ca(2+) influx and subsequent activation of stress-activated protein kinase pathways in osteoblasts. However, the activation mechanism and its significance in bone remodeling have not been fully elucidated. Here we show that TAK1 MAPKKK was activated by cyclic stretch loading of MC3T3-E1 cells. Knockdown of TAK1 attenuated the stretch-induced activation of JNK, p38, and NF-κB. Extracellular (EGTA) or intracellular (BAPTA/AM) Ca(2+) chelator prevented the stretch-induced activation of TAK1. Activation of TAK1 and its associated downstream signaling pathways were also suppressed by CaMKII inhibitors (KN-93 and KN-62). Furthermore, TAK1-mediated downstream pathways cooperatively induced the expression of IL-6 mRNA in the stretched MC3T3-E1 cells. We also confirmed that TAK1 mediates cyclic stretch-induced IL-6 protein synthesis in the cells using immunoblotting and ELISA. Finally, stretch loading of murine primary osteoblasts induced the expression of IL-6 mRNA via TAK1. Collectively, these data suggest that stretch-dependent Ca(2+) influx activates TAK1 via CaMKII, leading to the enhanced expression of IL-6 through JNK, p38, and NF-κB pathways in osteoblasts.

A mechanism for the suppression of interleukin-1-induced nuclear factor kappaB activation by protein phosphatase 2Ceta-2.

IL-1 (interleukin-1) is a pro-inflammatory cytokine that has a variety of effects during the process of inflammation. Stimulating cells with IL-1 initiates a signalling cascade that includes the activation of NF-kappaB (nuclear factor kappaB), and subsequently induces a variety of inflammatory genes. Although the molecular mechanism for the IL-1-induced activation of NF-kappaB has been well documented, much less is known about the mechanism by which protein phosphatases down-regulate this pathway. Here we show that mouse PP2Ceta-2 (protein serine/threonine phosphatase 2Ceta-2), a novel member of the protein serine/threonine phosphatase 2C family, inhibits the IL-1-NF-kappaB signalling pathway. Ectopic expression of PP2Ceta-2 in human embryonic kidney HEK293IL-1RI cells inhibited the IL-1-induced activation of NF-kappaB. TAK1 (transforming-growth-factor-beta-activated kinase 1) mediates the IL-1 signalling pathway to NF-kappaB, and we observed that the TAK1-induced activation of NF-kappaB was suppressed by PP2Ceta-2 expression. Expression of IKKbeta [IkappaB (inhibitory kappaB) kinase beta], which lies downstream of TAK1, activates NF-kappaB, and this activation was also readily reversed by PP2Ceta-2 co-expression. Additionally, PP2Ceta-2 knockdown with small interfering RNA further stimulated the IL-1-enhanced phosphorylation of IKKbeta and destabilization of IkappaBalpha in HeLa cells. PP2Ceta-2 knockdown also increased the IL-1-induced expression of IL-6 mRNA. Furthermore, IKKbeta was readily dephosphorylated by PP2Ceta-2 in vitro. These results suggest that PP2Ceta-2 inhibits the IL-1-NF-kappaB signalling pathway by selectively dephosphorylating IKKbeta.

Investigating bone morphogenetic protein (BMP) signaling in a newly established human cell line expressing BMP receptor type II.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor ß cytokine superfamily, elicit various biological effects in different tissues. BMP receptor type II (BMPRII) contains a unique carboxyl-terminal region that interacts with multiple signaling molecules. However, expression of endogenous BMPRII is low in various mammalian cell lines, which hampers the analysis of BMP signaling. Therefore, we established a human cell line expressing BMPRII tagged with a Flag epitope (BMPRII-Flag) using the tetracycline-controlled Flp-In T-REx gene expression system. The BMPRII-Flag gene was introduced into the Flp-In T-REx 293 (FT293) cell line, a derivative of human 293 embryonic kidney fibroblasts. Then we analyzed the expression of key BMP target genes, inhibitors of DNA binding (Id) family members (Id1, Id2, and Id3) and the inhibitory Smads Smad6 and Smad7, in parental FT293 cells and an established cell line, FT293-BMPRII, by quantitative real-time PCR. Tetracycline treatment significantly increased the expression of BMPRII-Flag mRNA and protein in FT293-BMPRII cells, but induced no significant changes in expression of Id1, Id2, Id3, Smad6, or Smad7 mRNA. In contrast, treatment with a BMPRII ligand BMP2 induced the expression of Id1, Id2, Id3, and Smad6 in parental FT293 cells and FT293-BMPRII cells. Tetracycline-induced BMPRII-Flag expression significantly enhanced the induction of Id1, Id3, and Smad6 mRNA expression in FT293-BMPRII cells treated with BMP2. These findings provide evidence that although BMPRII has no obvious effect on the expression of representative BMP target genes, it differentially modulates the responsiveness of target genes to BMP2.

Phosphorylation of protein phosphatase 2Czeta by c-Jun NH2-terminal kinase at Ser92 attenuates its phosphatase activity.

The protein phosphatase 2C (PP2C) family represents one of the four major protein Ser/Thr phosphatase activities in mammalian cells and contains at least 13 distinct gene products. Although PP2C family members regulate a variety of cellular functions, mechanisms of regulation of their activities are largely unknown. Here, we show that PP2Czeta, a PP2C family member that is enriched in testicular germ cells, is phosphorylated by c-Jun NH 2-terminal kinase (JNK) but not by p38 in vitro. Mass spectrometry and mutational analyses demonstrated that phosphorylation occurs at Ser (92), Thr (202), and Thr (205) of PP2Czeta. Phosphorylation of these Ser and Thr residues of PP2Czeta ectopically expressed in 293 cells was enhanced by osmotic stress and was attenuated by a JNK inhibitor but not by p38 or MEK inhibitors. Phosphorylation of PP2Czeta by TAK1-activated JNK repressed its phosphatase activity in cells, and alanine mutation at Ser (92) but not at Thr (202) or Thr (205) suppressed this inhibition. Taken together, these results suggest that specific phosphorylation of PP2Czeta at Ser (92) by stress-activated JNK attenuates its phosphatase activity in cells.

Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon.

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNFalpha (tumour necrosis factor alpha). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, ASK1 is transiently activated by autophosphorylation at Thr845. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr845. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.

Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation.

ISG15, an interferon-upregulated ubiquitin-like protein, is covalently conjugated to various cellular proteins (ISGylation). In this study, we found that protein phosphatase 2Cbeta (PP2Cbeta), which functions in the nuclear factor kappaB (NF-kappaB) pathway via dephosphorylation of TGF-beta-activated kinase, was ISGylated, and analysis by NF-kappaB luciferase reporter assay revealed that PP2Cbeta activity was suppressed by co-expression of ISG15, UBE1L, and UbcH8. We determined the ISGylation sites of PP2Cbeta and constructed its ISGylation-resistant mutant. In contrast to the wild type, this mutant suppressed the NF-kappaB pathway even in the presence of ISG15, UBE1L, and UbcH8. Thus, we propose that ISGylation negatively regulates PP2Cbeta activity.

Clathrin light chain b is capable of affecting potently a major protein phosphatase from microtubules (MT-PP1).

Clathrin light chain (CL) b purified from bovine brain postmicrotubule supernatant and identified by mass spectrometry potently inhibited a catalytic activity of a major protein phosphatase (PP) that was copurified with microtubules and recognized by antiPP1 antibodies. CLb similarly affected the catalytic subunit and holoenzyme of the PP, little inhibiting the activity of PP2A. Although the CLb from clathrin-coated vesicles was several hundredfold weaker than our purified CLb, the CLb in the postmicrotubule supernatant, independent of whether it was sedimentable or soluble, was as active as the purified CLb. Thus CLb may be a potent regulator of the PP.

Targeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain.

PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1lΔ/Δ mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.

Molecular cloning of PP2Ceta, a novel member of the protein phosphatase 2C family.

We have cloned a novel member of the mouse protein phosphatase 2C family, PP2Ceta. Sequence analysis suggests that PP2Ceta, PP2Czeta and NERPP-2C constitute a unique subgroup of the PP2C family. PP2Ceta had extremely low activity against alpha-casein compared with PP2Calpha and was localized mainly in cell nuclei, suggesting that PP2Ceta dephosphorylates a unique nuclear protein(s) in the cells.

Structural and functional characterization of mouse glutamate decarboxylase 67 gene promoter.

Neuronal expression of the mouse glutamate decarboxylase 67 (mGAD67) gene occurs exclusively in neurons that synthesize and release GABA (GABAergic neurons). This gene is also expressed in pancreatic islet cells and testicular spermatocytes. In order to elucidate the molecular mechanisms underlying the regulation of mGAD67 gene expression, we isolated and characterized the 5'-flanking region of this gene. Sequence analysis of a 10.2-kb DNA fragment of this gene containing a promoter region (8.4 kb) and noncoding exons 0A and 0B revealed the presence of numerous potential neuron-specific cis-regulatory elements. Functional analysis of the 5'-flanking region of exons 0A and 0B by transient transfection into cultured cells revealed that the region -98 to -52 close to exon 0A is important for the transcriptional activity of both exons 0A and 0B. In addition, we used transgenic mice to examine the expression pattern conferred by the 10.2 kb DNA fragment of the mGAD67 gene fused to the bacterial lacZ reporter gene. Transgene expression was observed in neurons of particular brain regions containing abundant GABAergic neurons such as the basal ganglia, in pancreatic islet cells and in testicular spermatocytes and spermatogonia. These results suggest that the 10.2 kb DNA fragment of the mGAD67 gene contains regulatory elements essential for its targeted expression in GABAergic neurons, islet cells and spermatocytes.

Classification of neural differentiation-associated genes in P19 embryonal carcinoma cells by their expression patterns induced after cell aggregation and/or retinoic acid treatment.

Expression of neural differentiation-associated genes was examined by RT-PCR and macroarray analyses during neural differentiation of P19 embryonal carcinoma cells induced by cell aggregation and/or retinoic acid (RA) treatment. Results revealed that the neural genes examined could be classified into 4 groups based on their expression patterns. The 1st group included the Wnt-1, Id-1, Id-3 and cdc42 genes, expression of which was altered by cell aggregation alone, but not by RA treatment alone. The 2nd group included the alphaN-catenin, Neuro D and GDNFRbeta genes, expression of which was altered by RA treatment alone, but not by cell aggregation. The 3rd group consisted of the Brn-2, TrkA, bcl-X, N-cadherin, E-cadherin and Otx-2 genes, expression of which was altered by either treatment. The 4th group included the ACTH, D4DR, NGC and Oct-3 genes, the expression of which changed only when both treatments were applied simultaneously. Expression of the Ets-1 and Fli-1 transcription factor genes was up-regulated by either treatment alone at initial stages of neural differentiation of P19 cells, although overexpression of these genes alone could not induce cell differentiation. Our results suggest that although both treatments are required for complete neural differentiation of P19 cells, cell aggregation or RA treatment alone drive differentiation to a certain extent at the gene expression level.

Activation mechanism of c-Jun amino-terminal kinase in the course of endodermal differentiation of P19 embryonic carcinoma cells.

c-Jun amino-terminal kinase (JNK) is known to be activated and play critical roles during neural and endodermal differentiation of P19 embryonic carcinoma cells. In this study we demonstrated that of the two upstream protein kinases of JNK, only MKK4 activity was substantially enhanced in the endodermally differentiating P19 cells. This enhanced activity of MKK4 stemmed from the increased expression of MKK4 and its activation by phosphorylation. Activated MKK4 and JNK were localized in both nucleus and cytoplasm of the differentiating cells, while they were localized only in the nucleus in the undifferentiated cells, suggesting multiple roles of JNK in the course of the endodermal differentiation of P19 cells.

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9.

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.

Acyl-CoA binding domain containing 3 (ACBD3) recruits the protein phosphatase PPM1L to ER-Golgi membrane contact sites.

The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER-Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER-Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER-Golgi interface.

Shigeru Tsuiki: a pioneer in the research fields of complex carbohydrates and protein phosphatases.

Dr Tsuiki made three major contributions during his illustrious career as a biochemist. First, he developed the procedure for mucin isolation from bovine submaxillary glands. His work became the basis for mucin biochemistry. Second, he identified four distinct molecular species of mammalian sialidase. Subsequent studies based on his work led to the discovery that sialidase plays a unique role as an intracellular signalling factor involved in the regulation of a variety of cellular functions. Finally, he established the molecular basis for the diversity of mammalian protein phosphatases through protein purification and molecular cloning. His work prompted the functional studies of protein phosphatases.