Vitamin D plays an important role in preserving healthy bones, and has additional roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development. Histone acetylation weakens the association of histones with DNA, and increases the accessibility of transcriptional regulatory proteins to chromatin templates, thereby increasing transcriptional activity of gene expression. Histone deacetylases remove the acetyl groups and condense chromatin structure, thereby preventing transcription. TSA is a potent histone deacetylase inhibitor and can significantly enhance gene expression. Bioactive food component, sulforaphane (SFN) is found in cruciferous vegetables and is known to be a histone deacetylase inhibitor, leading to transcriptional activation of gene expression. The objective of this study is to demonstrate that the bioactive food components modulate vitamin D action in adipocytes. To investigate the effects of TSA and SFN on vitamin D response, 3T3L1 mouse preadipocytes were treated with the combination of various concentrations of 1,25(OH)2 vitamin D, TSA, and SFN. Upon harvesting cells, the amounts of 24-hydroxylase mRNA, marker of vitamin D response, were measured by semiquantitative reverse transcriptase-PCR analysis. The results showed that the cells treated with 1μM TSA increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 3.5-fold (p < 0.05) at 1nM 1,25(OH)2 vitamin D and nearly 2.5-fold (p < 0.05) in 10 nM 1,25(OH)2 vitamin D, and the cells treated with 5μM SFN increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 1.4-fold at 1nM 1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D.

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Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório. / In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.