Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

c-Jun N-terminal kinase binding domain-dependent phosphorylation of mitogen-activated protein kinase kinase 4 and mitogen-activated protein kinase kinase 7 and balancing cross-talk between c-Jun N-terminal kinase and extracellular signal-regulated kinase pathways in cortical neurons

The c-Jun N-terminal kinase (JNK) is a mitogen-activated protein kinase (MAPK) activated by stress-signals and involved in many different diseases. Previous results proved the powerful effect of the cell permeable peptide inhibitor d-JNKI1 (d-retro-inverso form of c-Jun N-terminal kinase-inhibitor) against neuronal death in CNS diseases, but the precise features of this neuroprotection remain unclear. We here performed cell-free and in vitro experiments for a deeper characterization of d-JNKI1 features in physiological conditions. This peptide works by preventing JNK interaction with its c-Jun N-terminal kinase-binding domain (JBD) dependent targets. We here focused on the two JNK upstream MAPKKs, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7), because they contain a JBD homology domain. We proved that d-JNKI1 prevents MKK4 and MKK7 activity in cell-free and in vitro experiments: these MAPKK could be considered not only activators but also substrates of JNK. This means that d-JNKI1 can interrupt downstream but also upstream events along the JNK cascade, highlighting a new remarkable feature of this peptide. We also showed the lack of any direct effect of the peptide on p38, MEK1, and extracellular signal-regulated kinase (ERK) in cell free, while in rat primary cortical neurons JNK inhibition activates the MEK1-ERK-Ets1/c-Fos cascade. JNK inhibition induces a compensatory effect and leads to ERK activation via MEK1, resulting in an activation of the survival pathway-(MEK1/ERK) as a consequence of the death pathway-(JNK) inhibition. This study should hold as an important step to clarify the strong neuroprotective effect of d-JNKI1.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Calmodulin-dependent protein kinase IV during T-cell development.

In a primary cell culture system of fetal rat brain, the calmodulin-dependent protein-kinase IV (CaMKIV) could be induced by the thyroid hormone T3 in a time- and concentration-dependent manner, provided the tissue was excised not later than day 15 of gestation (E15) (Krebs et al., J. Biol. Chem. 271, 11055, 1996). We report here that in the fetal thymus CaMKIV could not be detected earlier than day 16 of gestation and that the expression of this enzyme was fully upregulated at day 18. In mouse fetal thymus organ culture (FTOC) of day 14 embryonic thymus, CaMKIV could not be detected, even after several days of culture if a minimal culture medium lacking fetal calf serum was used. However, after addition of fetal calf serum to the culture medium the expression of CaMKIV could be specifically induced. Furthermore, it could also be shown that during T-cell development in the adult murine thymus the expression of CaMKIV was tightly regulated. Taken together, these results demonstrate that the expression of CaMKIV, an enzyme involved in the regulation of Ca(2+)-dependent gene expression, is itself under stringent regulatory control during tissue development.

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Expression, purification and biochemical characterization of recombinant Ca-dependent protein kinase 2 of the malaria parasite Plasmodium falciparum.

Calcium-dependent protein kinases (CDPKs) are serine/threonine kinases that react in response to calcium which functions as a trigger for several mechanisms in plants and invertebrates, but not in mammals. Recent structural studies have defined the role of calcium in the activation of CDPKs and have elucidated the important structural changes caused by calcium in order to allow the kinase domain of CDPK to bind and phosphorylate the substrate. However, the role of autophosphorylation in CDPKs is still not fully understood. In Plasmodium falciparum, seven CDPKs have been identified by sequence comparison, and four of them have been characterized and assigned to play a role in parasite motility, gametogenesis and egress from red blood cells. Although PfCDPK2 was already discovered in 1997, little is known about this enzyme and its metabolic role. In this work, we have expressed and purified PfCDPK2 at high purity in its unphosphorylated form and characterized its biochemical properties. Moreover, propositions about putative substrates in P. falciparum are made based on the analysis of the phosphorylation sites on the artificial substrate myelin basic protein (MBP).

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Hepatitis B Virus e Antigen Physically Associates With Receptor-Interacting Serine/Threonine Protein Kinase 2 and Regulates IL-6 Gene Expression.

We previously reported that hepatitis B virus (HBV) e antigen (HBeAg) inhibits production of interleukin 6 by suppressing NF-κB activation. NF-κB is known to be activated through receptor-interacting serine/threonine protein kinase 2 (RIPK2), and we examined the mechanisms of interleukin 6 regulation by HBeAg. HBeAg inhibits RIPK2 expression and interacts with RIPK2, which may represent 2 mechanisms through which HBeAg blocks nucleotide-binding oligomerization domain-containing protein 1 ligand-induced NF-κB activation in HepG2 cells. Our findings identified novel molecular mechanisms whereby HBeAg modulates intracellular signaling pathways by targeting RIPK2, supporting the concept that HBeAg could impair both innate and adaptive immune responses to promote chronic HBV infection.

Calcineurin blockade prevents cardiac mitogen-activated protein kinase activation and hypertrophy in renovascular hypertension.

Chronic stimulation of the renin-angiotensin system induces an elevation of blood pressure and the development of cardiac hypertrophy via the actions of its effector, angiotensin II. In cardiomyocytes, mitogen-activated protein kinases as well as protein kinase C isoforms have been shown to be important in the transduction of trophic signals. The Ca(2+)/calmodulin-dependent phosphatase calcineurin has also been suggested to play a role in cardiac growth. In the present report, we investigate possible cross-talks between calcineurin, protein kinase C, and mitogen-activated protein kinase pathways in controlling angiotensin II-induced hypertrophy. Angiotensin II-stimulated cardiomyocytes and mice with angiotensin II-dependent renovascular hypertension were treated with the calcineurin inhibitor cyclosporin A. Calcineurin, protein kinase C, and mitogen-activated protein kinase activations were determined. We show that cyclosporin A blocks angiotensin II-induced mitogen-activated protein kinase activation in cultured primary cardiomyocytes and in the heart of hypertensive mice. Cyclosporin A also inhibits specific protein kinase C isoforms. In vivo, cyclosporin A prevents the development of cardiac hypertrophy, and this effect appears to be independent of hemodynamic changes. These data suggest cross-talks between the calcineurin pathway, the protein kinase C, and the mitogen-activated protein kinase signaling cascades in transducing angiotensin II-mediated stimuli in cardiomyocytes and could provide the basis for an integrated model of cardiac hypertrophy.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2.

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.