RESUMO
INTRODUCTION: Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17ß-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue. METHODS: We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes. RESULTS: Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⻹¹ M to 10â»7M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-ß. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole. CONCLUSIONS: We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-ß. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.
Assuntos
Aromatase/biossíntese , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Estrogênios/metabolismo , Osteoartrite/metabolismo , Adulto , Idoso , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
To analyse the hypothesis as to whether there is a functional relationship between human cationic amino acid transporters (hCATs, y(+) transporter, the main transporter of L-arginine and L-lysine) and human ß-defensin (important components of immune function) production on the ocular surface, arginase and nitrate monoxide synthase (NOS), enzymes that compete for L-arginine, were inhibited by norNOHA (N(omega)-hydroxy-nor-L-arginine) and/or L-NAME (NG-nitro-L-arginine methyl ester) in cultured human corneal epithelial cells. In addition, the transport activity of hCAT proteins was inhibited or activated through α-tocopherol or PMA (phorbol myristate acetate), respectively. Concentrations of the human inducible ß-defensins (hBD) 2 and 3 were determined by ELISA experiments. The basic expression of hBD3 in non-stimulated HCE cells significantly exceeded that of hBD2. Both ß-defensins also differed as to how readily their excretion could be stimulated. HBD2 excretion rate was 3.5 time more by L-NAME, whereas norNOHA had no effect. In contrast, hBD3 excretion was increased by norNOHA by a factor of 1.5 but L-NAME alone had no effect. The excretion of both ß-defensins was increased 3- and 6-fold by combined administration of L-NAME, norNOHA and interleukin (IL)-1ß. Administration of α-tocopherol increased hBD2 excretion twofold. No effect was observed for hBD3. With PMA, on the other hand, a reduction in secretion for both ß-defensins was observed. These in vitro findings provide evidence of a functional association between CAT proteins and ß-defensins 2 and 3 opening up a new field of research with pharmacological perspectives for treatment of inflammatory diseases such as keratitis or dry eye disease.