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HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

View Article: PubMed Central - PubMed

ABSTRACT

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

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Expression of PPP1R14a is regulated by HMGB4.(a) Inhibition of PPP1R14a expression in HEK 293T -cells constitutively expressing HMGB4-EGFP. Three independent repeats were analyzed by comparing representative EGFP expressing control and HMGB4-EGFP expressing cells. Results from representative analysis are shown. (b) Mouse E14 cortical neurons and C2C12 myoblast cells, both expressing endogenous HMGB4 mRNA, were treated with HMGB4 Vivo-Morpholino to downregulate translation of HMGB4. The expression levels of PPP1R14a were analyzed with qPCR. The levels of the PPP1R14a transcript in HMGB4 Vivo-Morpholino -treated cells were normalized to the PPP1R14a transcript levels in control Vivo-Morpholino -treated cells (*p < 0.05, ± SEM; n = 6 in C2C12 experiment; n = 18 in neuron experiment).
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f5: Expression of PPP1R14a is regulated by HMGB4.(a) Inhibition of PPP1R14a expression in HEK 293T -cells constitutively expressing HMGB4-EGFP. Three independent repeats were analyzed by comparing representative EGFP expressing control and HMGB4-EGFP expressing cells. Results from representative analysis are shown. (b) Mouse E14 cortical neurons and C2C12 myoblast cells, both expressing endogenous HMGB4 mRNA, were treated with HMGB4 Vivo-Morpholino to downregulate translation of HMGB4. The expression levels of PPP1R14a were analyzed with qPCR. The levels of the PPP1R14a transcript in HMGB4 Vivo-Morpholino -treated cells were normalized to the PPP1R14a transcript levels in control Vivo-Morpholino -treated cells (*p < 0.05, ± SEM; n = 6 in C2C12 experiment; n = 18 in neuron experiment).

Mentions: The most significant change in gene expression, induced by HMGB4-EGFP in microarray analyses was seen in the expression level of the PPP1R14a gene, which was downregulated approximately 140 –fold (Supplementary Table S2). PPP1R14a codes for protein phosphatase 1 regulatory subunit 14A (also known as C-kinase activated Protein phosphatase-1 Inhibitor of 17 kDa, CPI-17) that regulates multiple cellular functions, including cell adhesion, tumorigenesis and brain development. PPP1R14a downregulation in HMGB4-EGFP expressing cell clones was further evaluated by qPCR (Fig. 5a). To confirm that HMGB4 directly regulates the expression of the PPP1R14a gene in transformed cells we knocked down endogenous HMGB4 translation in mouse C2C12 myoblast cells (Fig. 5b). When these cells were treated with HMGB4 Vivo-Morpholinos, they expressed higher levels of PPP1R14a mRNA than the cells treated with standard control Vivo-Morpholinos. As our microarray data revealed, cell adhesion processes were most significantly affected in HMGB4 –overexpressing cells (Table 1). Knock down experiments suggested the possibility that one relevant HMGB4 regulated mechanism in cells is PPP1R14a-dependent regulation of phosphorylation of cell adhesion controlling proteins. One such protein is merlin, when phosphorylated, is a key regulator of adhesion in many cell types24252627. Therefore we tested merlin phosphorylation in HMGB4 overexpressing cells. The relative phosphorylation state of merlin remained unaltered in cell cultures expressing HMGB4-EGFP when compared to control EGFP –expressing cell cultures (0.60 ± 0.31 and 1.00 ± 0.08, respectively). This suggests that other processes than merlin phosphorylation dependent cell adhesion are relevant in HMGB4 mediated regulation of PPP1R14a.


HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation
Expression of PPP1R14a is regulated by HMGB4.(a) Inhibition of PPP1R14a expression in HEK 293T -cells constitutively expressing HMGB4-EGFP. Three independent repeats were analyzed by comparing representative EGFP expressing control and HMGB4-EGFP expressing cells. Results from representative analysis are shown. (b) Mouse E14 cortical neurons and C2C12 myoblast cells, both expressing endogenous HMGB4 mRNA, were treated with HMGB4 Vivo-Morpholino to downregulate translation of HMGB4. The expression levels of PPP1R14a were analyzed with qPCR. The levels of the PPP1R14a transcript in HMGB4 Vivo-Morpholino -treated cells were normalized to the PPP1R14a transcript levels in control Vivo-Morpholino -treated cells (*p < 0.05, ± SEM; n = 6 in C2C12 experiment; n = 18 in neuron experiment).
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f5: Expression of PPP1R14a is regulated by HMGB4.(a) Inhibition of PPP1R14a expression in HEK 293T -cells constitutively expressing HMGB4-EGFP. Three independent repeats were analyzed by comparing representative EGFP expressing control and HMGB4-EGFP expressing cells. Results from representative analysis are shown. (b) Mouse E14 cortical neurons and C2C12 myoblast cells, both expressing endogenous HMGB4 mRNA, were treated with HMGB4 Vivo-Morpholino to downregulate translation of HMGB4. The expression levels of PPP1R14a were analyzed with qPCR. The levels of the PPP1R14a transcript in HMGB4 Vivo-Morpholino -treated cells were normalized to the PPP1R14a transcript levels in control Vivo-Morpholino -treated cells (*p < 0.05, ± SEM; n = 6 in C2C12 experiment; n = 18 in neuron experiment).
Mentions: The most significant change in gene expression, induced by HMGB4-EGFP in microarray analyses was seen in the expression level of the PPP1R14a gene, which was downregulated approximately 140 –fold (Supplementary Table S2). PPP1R14a codes for protein phosphatase 1 regulatory subunit 14A (also known as C-kinase activated Protein phosphatase-1 Inhibitor of 17 kDa, CPI-17) that regulates multiple cellular functions, including cell adhesion, tumorigenesis and brain development. PPP1R14a downregulation in HMGB4-EGFP expressing cell clones was further evaluated by qPCR (Fig. 5a). To confirm that HMGB4 directly regulates the expression of the PPP1R14a gene in transformed cells we knocked down endogenous HMGB4 translation in mouse C2C12 myoblast cells (Fig. 5b). When these cells were treated with HMGB4 Vivo-Morpholinos, they expressed higher levels of PPP1R14a mRNA than the cells treated with standard control Vivo-Morpholinos. As our microarray data revealed, cell adhesion processes were most significantly affected in HMGB4 –overexpressing cells (Table 1). Knock down experiments suggested the possibility that one relevant HMGB4 regulated mechanism in cells is PPP1R14a-dependent regulation of phosphorylation of cell adhesion controlling proteins. One such protein is merlin, when phosphorylated, is a key regulator of adhesion in many cell types24252627. Therefore we tested merlin phosphorylation in HMGB4 overexpressing cells. The relative phosphorylation state of merlin remained unaltered in cell cultures expressing HMGB4-EGFP when compared to control EGFP –expressing cell cultures (0.60 ± 0.31 and 1.00 ± 0.08, respectively). This suggests that other processes than merlin phosphorylation dependent cell adhesion are relevant in HMGB4 mediated regulation of PPP1R14a.

View Article: PubMed Central - PubMed

ABSTRACT

HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value &le;0.013 (n&thinsp;=&thinsp;3) in a microarray analysis and displayed strongest association with adhesion and histone H2A &ndash;processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

No MeSH data available.


Related in: MedlinePlus