No abstract

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 are the enzymes involved in melanin biosynthesis and are preferentially expressed in pigment cells. Their human gene promoters share the 11-base pair M box containing a CATGTG motif, which was shown here to be bound in vitro by microphthalmia-associated transcription factor (MITF). Transient cotransfection analysis showed that MITF overexpression increased the expression of a reporter gene under the control of the human tyrosinase or TRP-1 gene promoter but not the TRP-2 promoter. The promoter activation caused by MITF is dependent on each CATGTG motif of the distal enhancer element, the M box, and the initiator E box of the tyrosinase gene and the TRP-1 M box. Furthermore, a truncated MITF lacking the carboxyl-terminal 125 amino acid residues transactivated the tyrosinase promoter less efficiently than did MITF, suggesting that MITF's carboxyl terminus contains a transcriptional activation domain, but unexpectedly such a truncated MITF remarkably transactivated the TRP-2 gene promoter. These results suggest that MITF is sufficient to direct pigment cell-specific transcription of the tyrosinase and TRP-1 genes but not the TRP-2 gene.

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

Plasma concentrations of endothelin, a vasoconstrictor peptide released from vascular endothelial cells, have been measured by radioimmunoassay in 100 patients with diabetes mellitus and 19 healthy subjects. The plasma immunoreactive-endothelin concentrations were found to be greatly raised in the patients with diabetes (1,880 +/- 120 fmol/l, mean +/- SEM) compared with the healthy subjects (540 +/- 50 fmol/l, p less than 0.005). The elevation of immunoreactive-endothelin could not be explained by secondary changes in blood pressure or renal disease and did not correlate with the presence of diabetic retinopathy, duration of diabetes mellitus, fasting blood glucose or serum fructosamine. Fast protein liquid chromatographic analysis of the diabetic plasma immunoreactive-endothelin showed three forms, one in a very big molecular weight position, one intermediate and one in the position of endothelin-1 itself. No material appeared in the positions of endothelin-2 and 3. Chromatographic analysis of normal plasma showed only the big molecular weight peak while material in the endothelin-1, 2 or 3 positions was below detection. The elevation of endothelin in diabetic patients may be a marker of, and further exacerbate, their vascular disease.

Heme oxygenase (EC 1.14.99.3) is the rate-limiting enzyme in heme catabolism that cleaves heme at the ␣-methene bridge to form biliverdin IX␣, carbon monoxide, and iron (1, 2). Biliverdin IX␣ is immediately converted by biliverdin reductase to bilirubin IX␣ that is transported to the liver for conjugation and excretion into bile (3). There are two isozymes of heme oxygenase, heme oxygenase-1 (HO-1) 1 and heme oxygenase-2 (HO-2) (4, 5). HO-1 is inducible whereas HO-2 is constitutively expressed in human cells (6). Expression of HO-1 mRNA is highly increased in human cells by the substrate heme (7), heavy metals (8, 9), UV irradiation (10), and nitric oxide donors (11)(12)(13)(14). Because bilirubin IX␣ functions as a natural radical scavenger (15, 16), induction of HO-1 probably represents a protective response against oxidative stress. The physiological importance of HO-1 has been confirmed by the phenotypic consequences of the HO-1-deficient mice (17) and a patient with HO-1 deficiency (18).Induction of HO-1 has been extensively studied for the last few decades by many investigators. In contrast, repression of HO-1 expression has been largely ignored, despite its physiological importance (3). We have shown that HO-1 is not induced or rather reduced by heat shock in human cells (19), whereas rat HO-1 is a heat shock protein (20,21). The expression levels of HO-1 are also decreased in human glioblastoma cells by the treatment with interferon-␥ (22). In addition, hypoxia represses HO-1 mRNA expression in primary cultures of human umbilical vein endothelial cells (HUVECs), human astrocytes, and human coronary arterial endothelial cells (23). On the other hand, hypoxia increased HO-1 expression in rat liver (24) and heart (25) and in various cultured animal cells, including Chinese hamster ovary cells (26), rat ventricular smooth muscle cells (27,28), and rat myocytes (29). These results suggest the inter-species difference in the regulation of HO-1 gene expression by hypoxia between human and animal cells.The inter-species variations in the hypoxic response are of clinical significance because hypoxia is involved in the pathophysiology of various disorders, including ischemic heart disease, cerebrovascular disease, cancer, sleep apnea syndrome, and chronic obstructive pulmonary disease, which account for common causes of death and disability in the developed world. Mammalian cells respond to hypoxia in part by increased expression of several genes coding for erythropoietin (30), vascular endothelial growth factor (31), adrenomedullin (32, 33), and glycolytic enzymes (34,35), all of which cooperate to protect cells and tissues against the hypoxic state. Hypoxia-inducible