High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo.

High mobility group proteins are chromatin binding factors with key roles in maintenance of nuclear homeostasis. The evidence indicates that extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promoting inflammation and participating to the pathogenesis of several disorders in peripheral organs. In this study, we have investigated the expression levels and relocation dynamics of HMGB1 in neural cells, as well as its neuropathological potential. We report that HMGB1 is released in the culture media of neurons and astrocytes challenged with necrotic but not apoptotic stimuli. Recombinant HMGB1 prompts induction of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2, interleukin-1beta, and tumor necrosis factor alpha, and increases excitotoxic as well as ischemic neuronal death in vitro. Dexamethasone reduces HMGB1 dependent immune glia activation, having no effect on the protein's neurotoxic effects. HMGB1 is expressed in the nucleus of neurons and astrocytes of the mouse brain, and promptly (1 h) translocates into the cytoplasm of neurons within the ischemic brain. Brain microinjection of HMGB1 increases the transcript levels of pro-inflammatory mediators and sensitizes the tissue to the ischemic injury. Together, data underscore the neuropathological role of nuclear HMGB1, and point to the protein as a mediator of post-ischemic brain damage.

Extracellular processing of amphoterin generates a peptide active on erythroleukaemia cell differentiation.

The release of amphoterin by murine erythroleukaemia cells exposed to the chemical inducer hexamethylenebisacetamide represents an essential step for the process of their terminal differentiation. Once exported in the culture medium, amphoterin undergoes limited proteolysis, catalysed by a serine proteinase also secreted by stimulated cells. The isolated proteinase is responsible for degradation of amphoterin, with the production of a 10-amino-acid-residue fragment, specifically retaining the cell-differentiation-stimulating activity of the native protein molecule. This peptide does not express other properties of amphoterin, such as protein kinase C-stimulating activity or systemic toxicity. These findings define a selective mechanism accounting for extracellular amphoterin functional maturation.

Human neuroblastoma cell differentiation requires protein kinase C-theta.

Neuroblastoma LAN-5 cells exposed to retinoic acid cease to multiply and extend neurite outgrowths acquiring a neuronal phenotype. We now report that protein kinase C-theta; (PKC-theta;) isozyme is involved in this differentiation process due to the following findings: (i) PKC-theta; is expressed by LAN-5 cells as a nuclear and perinuclear protein; (ii) cell stimulation with retinoic acid promotes in a large increase in the expression level of the kinase and its intracellular redistribution; and (iii) a PKC-theta; antisense oligonucleotide reduces at the same time the expression level of the kinase and the cell response to retinoic acid. Altogether these data are consistent with a specific role played by PKC-theta; in the differentiation program of neuronal cells.

Neuronal differentiation of PC12 cells involves changes in protein kinase C-theta distribution and molecular properties.

In this study we demonstrate that the rat pheochromocytoma PC12 cell line expresses the novel protein kinase C isozyme designated PKC-θ. The isozyme is almost completely localized in the nuclear compartment of proliferating cells. Following stimulation with the nerve growth factor, PKC-θ is redistributed into the cytoplasm and the outgrowing neurite processes, mostly as a cytoskeletal associated kinase. This event is accompanied by an eightfold increase in the expression level and by the appearance of specific modifications of PKC-θ molecule. Conversely, the kinase is down-regulated once cells reach the terminally differentiated state displaying a neuron-like phenotype. These data suggest a functional role for the kinase in the regulation of cytoskeletal modeling along the multistage differentiation process of PC12 cells.

Acyl-CoA-binding protein is a potent m-calpain activator.

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

Protein kinase C-theta is specifically activated in murine erythroleukaemia cells during mitosis.

Protein kinase C-theta is a member of the n-protein kinase C subfamily that in mitotic cells translocates to centrosomes and kinetochores. Although this kinase is expressed in comparable amounts in murine erythroleukaemia cells during the interphase or metaphase, when localized in the mitotic structures, it selectively phosphorylates a 66 kDa protein, also associated to chromosomes. Moreover, protein kinase C-theta immunoprecipitated from cells at the metaphase results four times more active in the absence of lipid cofactors as compared with the kinase obtained from cells in the interphase. This activation is accomplished by interaction of protein kinase C-theta with a protein factor which also promotes an increased autophosphorylation of the kinase. These findings indicate that in the mitotic phase of the cell cycle, protein kinase C-theta recognizes a protein factor which operates as a positive modulator of the kinase activity in the absence lipids.

Protein kinase C-theta is specifically localized on centrosomes and kinetochores in mitotic cells.

In this study we provide evidence that the protein kinase C (PKC)-straight theta isoenzyme is recruited on to the mitotic spindle in dividing murine erythroleukaemia (MEL) cells and associates specifically with centrosome and kinetochore structures. None of the other PKC isoenzymes (-alpha, -delta, -epsilon, -mu and -zeta) expressed by MEL cells shows this localization on the mitotic spindle. An identical subcellular distribution of PKC-straight theta is also observed in dividing murine P3 myeloma cells and human LAN-5 neuroblastoma cells, indicating that this PKC isoenzyme interacts with the mitotic apparatus in mammalian cells. In phorbol-ester-treated non-growing MEL cells, a rapid change in the intracellular distribution of PKC-straight theta occurs. Under these conditions, PKC-straight theta is translocated from the nuclear to the cytosolic cell compartment, an event that is accompanied by phosphorylation of the PKC-straight theta molecule and is followed by its down-regulation. The recovery of cell growth capacity results in the concomitant reappearance of PKC-straight theta. Furthermore, when MEL cells acquire the differentiated non-growing phenotype, the level of PKC-straight theta is reduced to less than 5%, suggesting that this PKC isoenzyme is no longer required. We propose that, unlike other members of the PKC family, PKC-straight theta may play a role in cell proliferation.

Mechanism of action of a new component of the Ca(2+)-dependent proteolytic system in rat brain: the calpain activator.

Rat brain contains a calpain activator specific for the mu-form of the proteinase. We now report that this protein factor binds to the catalytic 80 kDa calpain subunit, promoting the dissociation of the heterodimer structure of the proteinase. The successive steps of the activation process, namely the two autoproteolytic steps producing the 78 kDa and the 75 kDa calpain forms, result in a 100 times faster rate. The activator competes with calpastatin and associates with the inner surface of plasma membranes. Based on its properties, the calpain activator can be visualised as the molecule indicating the sites for calpain activation at which the proteinase can also elude the negative control exerted by calpastatin.

Properties of calpastatin forms in rat brain.

Four recombinant calpastatin forms, deduced from rat brain mRNAs and differing in the number of inhibitory repetitive domains from zero to four, were expressed and characterized for their inhibitory efficiency on mu- and m-calpain. Although the most effective one is a truncated calpastatin form composed of the N-terminal region (domain L) and a single inhibitory domain, all inhibitors are more active against mu-calpain, but are preferentially degraded and inactivated by m-calpain. The protein form composed exclusively of a domain L is deprived of any inhibitory activity but prevents inhibition of calpain by the other calpastatin forms, indicating that this calpastatin region could be relevant in the recognition of the proteinase. A calpastatin form having molecular properties similar to those of the recombinant truncated calpastatin, has also been found in rat brain. It does not derive from proteolysis of a higher molecular mass precursor. The expression of multiple calpastatin forms may be relevant for the specific modulation of the different calpain isozymes normally present in a single cell type.

Rat brain contains multiple mRNAs for calpastatin.

This work was undertaken to establish the forms of the calpain inhibitor, calpastatin, expressed in the brain tissue. Five cDNA clones were obtained and the corresponding amino acid sequences were deduced. Three of these proteins contain an N-terminal domain (domain L) and four inhibitory repeats typical of the calpastatin molecule. The other two are truncated forms, containing the domain L, free or associated with a single inhibitory repeat. Other differences, due to exon skipping, produce calpastatin forms with different susceptibility to posttranslational modifications. The more represented mRNA form corresponds to a calpastatin molecule containing the four inhibitory domains. These results may be useful to understand the involvement of calpain in the onset of acute and degenerative disorders of the central nervous system.

Stimulated astrocytes release high-mobility group 1 protein, an inducer of LAN-5 neuroblastoma cell differentiation.

Stimulated astrocytes specifically release large amounts of high-mobility group 1 protein into the extracellular medium. The identity of the released protein has been established on the basis of its biological activity on murine erythroleukaemia cells and by its immunoreactivity against a specific monoclonal antibody. High-mobility group 1 protein also plays an essential role in differentiation of LAN-5 neuroblastoma cells which, following stimulation with retinoic acid, express high-mobility group 1 protein on to the external surface of the plasma membrane. In retinoic acid-induced LAN-5 cells, high-mobility group 1 protein is not secreted but is accumulated in a membrane-bound form, particularly at the level of neurite outgrowths. These cells can also be induced to differentiate by high-mobility group 1 protein coated on the surface of the cell culture vessels. The specific function of the protein in this process is indicated by inhibition of cell differentiation by an anti-high-mobility group 1 protein antibody. The data are consistent with a role of high-mobility group 1 protein in promoting cell-cell interactions and in the development of nerve tissues.

Secretion and binding of HMG1 protein to the external surface of the membrane are required for murine erythroleukemia cell differentiation.

We show here that murine erythroleukemia (MEL) cells, following induction with hexamethylene bisacetamide, accumulate high mobility group (HMG)1 protein onto the external surface of the cell in a membrane-associated form detectable by immunostaining with a specific anti-HMG1 protein antibody. This association is maximal at a time corresponding to cell commitment. At longer times, immunostainable cells are progressively reduced and become almost completely undetectable along with the appearance of hemoglobin molecules. Binding to MEL cells does not affect the native molecular structure of HMG1 protein. The type of functional correlation between HMG1 protein and MEL cell differentiation is suggested by the observation that if an anti-HMG1 protein antibody is added at the same time of the inducer almost complete inhibition of cell differentiation is observed, whereas if the antibody is added within the time period in which cells undergo through irreversible commitment, inhibition progressively disappears. A correlation between MEL cell commitment and the biological effect of HMG1 protein can thus be consistently suggested.

Extracellular high-mobility group 1 protein is essential for murine erythroleukaemia cell differentiation.

A high-mobility group 1 (HMG1) protein type isolated from murine erythroleukaemia (MEL) cells promotes acceleration of the differentiation process when added to a MEL cell culture together with the inducer hexamethylene bisacetamide. We now provide direct evidence that the presence of HMG1 protein in the extracellular medium is essential for terminal erythroid differentiation. An extracellular function for HMG1 protein in MEL cell is further supported by a demonstration that this protein is released from MEL cells exposed to the chemical inducer and that the addition of an anti-(HMG1 protein) monoclonal antibody to the cell culture inhibits the differentiation process almost completely. The release of HMG1 protein from MEL cells is modulated by compounds affecting cell calcium homoeostasis, such as a calcium ionophore or verapamil. In fact, in the presence of the ionophore an increased rate of differentiation is accompanied by an enhanced extracellular release of HMG1 protein, whereas in the presence of verapamil both phenomena are significantly decreased.

A 6 kDa protein homologous to the N-terminus of the HMG1 protein promoting stimulation of murine erythroleukemia cell differentiation.

Murine erythroleukemia (MEL) cells, in addition to an mRNA coding for a 30 kDa high mobility group (HMG)-1 protein, contain an mRNA coding for a 6 kDa HMG1 protein having the following structural properties: (1) its primary structure has 90% homology with the N-terminal sequence of the 30 kDa HMG1 protein; (2) it contains a consensus region of the HMG1 protein family; (3) it is deprived of the cluster of acidic amino acids that characterizes the C-terminal region of the 30 kDa HMG1 protein. This novel small Mr HMG1 protein has been expressed in prokaryotic cells and tested to establish similarities and differences in activity compared to the homologous higher Mr HMG1 protein. It has been found that the low Mr HMG1 form is not released from MEL cells following induction to erythroid differentiation, but is still effective, although with much less efficiency, when added to the external medium, in promoting acceleration in the rate of MEL cell differentiation as well as in activation of alpha-protein kinase C. Altogether these results provide evidence for the presence in MEL cells of a multigene family that encodes at least two different HMG1-type sequences most presumably involved, at distinct cellular sites, in different functions although commonly related to the promotion of cell differentiation. Additional information can be considered concerning the relationship between the characteristic N-terminal sequence of HMG1 protein and the extracellular activity on MEL cell differentiation.

Correlation between levels of delta protein kinase C and resistance to differentiation in murine erythroleukemia cells.

It has been demonstrated that the level of delta protein kinase C is inversely correlated to the responsiveness of murine erythroleukemia cells to chemical induction to terminal erythroid differentiation. In these cells, deltaPKC is largely present in a membrane associated form, and thus in a constitutively active state, a condition which characterizes the undifferentiated phenotype. Accordingly, commitment to cell differentiation has been shown to be preceded by down regulation of deltaPKC, a process significantly accelerated and induced to almost completion by the differentiation enhancing factor (DEF) in a dose dependent manner. The present results provide a better understanding of the role of deltaPKC in characterizing the undifferentiated MEL cell phenotype and suggest a relationship between the acceleration in the rate of differentiation induced by DEF and the down regulation of this kinase form.

Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells.

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells.

Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity.

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

Extracellular release of the 'differentiation enhancing factor', a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation.

Differentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as 'amphoterin' on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N-terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation.

Identity in molecular structure between "differentiation enhancing factor" of murine erythroleukemia cells and the 30 kD heparin-binding protein of developing rat brain.

A 29 kD protein previously isolated from murine erythroleukemia (MEL) cells and shown to enhance the rate of differentiation of these cells has now been demonstrated to possess an amino acid sequence identical to that reported for the 30 kD heparin-binding protein from developing rat brain, named amphoterin after its highly dipolar structure. The identity between the two proteins has been established on the basis of a strong heparin binding affinity and a complete homology in the amino acid sequences of N-terminal region as well as of several tryptic peptides. Furthermore, the cDNA encoding this protein has been isolated from MEL cell mRNA, by means of reverse transcriptase-polymerase chain reaction, and its sequence was found to correspond to that of amphoterin. The MEL cell differentiation enhancing factor, previously abbreviated as DEF, is again confirmed to reduce the latent period preceding the appearance of hexamethylenebisacetamide induced cell commitment and to stimulate the catalytic activity of alpha-protein kinase C. Thus, here we demonstrate that a protein expressed in MEL cells, whose sequence is identical to that previously reported for amphoterin, plays an essential role in promoting cell differentiation, thereby indicating a new relevant function of amphoterin.