Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-16
2004-06-22
Leffers, Gerry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S029000, C435S455000, C536S023100
Reexamination Certificate
active
06753140
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a positive cis-regulatory (enhancer) element of human &bgr;
3
-adrenergic receptor responsible for its transcription in SK-N-MC cells. Such element is composed of three DNA binding sites that act synergistically and is located 6.5 kb upstream from the translation start site of the &bgr;
3
-adrenergic receptor. The invention further relates to use of this enhancer element for regulated gene expression and for drug screening.
BACKGROUND OF THE INVENTION
The &bgr;
3
-adrenergic receptor (&bgr;
3
-AR) is an important regulator of metabolic activity in brown and white adipose tissue, two major sites for regulation of energy balance. The &bgr;
3
-AR belongs to a family of G-protein coupled receptors. Its binding to endogenous ligand or specific synthetic agonist leads to activation of adenylate cyclase, an increased concentration of cAMP, and an increased activity of PKA, resulting in increased thernogenic activity and heat production in brown adipose tissue (BAT) and lipolysis in white adipose tissue (WAT). Since &bgr;
3
-AR stimulation causes an increase in thermogenic activity and a less efficient utilization of metabolic fuels, its sustained activation should be of benefit in the treatment of obesity, and improvement of glycemic control in type II diabetes. Indeed, numerous reports have shown that stimulation of &bgr;
3
-ARs cause weight loss and improvement in glycemic control in rodent models of these diseases (Carroll et al., Diabetes 34:1198-1204, 1984; Umekawa et al., European Journal of Endo.136:429-437, 1997; Yoshida et al, J. Nutr. Sci. Vitaminol (Tokyo) 36:75-80, 1990; Yoshida et al., Endocrinol Japon 38:397-403, 1991; Smith et al., New Antidiabetic Drugs, Ed.: Bailey, C. J., and Flatt, P. R., London, 1990; Largis et al., Drug Dev. Res. 32:69-76, 1994; Bloom et al., Journal of Medicinal Chemistry 35:3081-3084, 1992; Cawthorne et al., American Journal of Clinical Nutrition 55:252S-257S, 1992).
Despite the well characterized role of &bgr;
3
-ARs in rodents, its role in the regulation of energy balance in man is still not clear. Observations by several groups of investigators (Walston et al., New Engl. J. Med. 333:343-347, 1995; Kadowaki et al., Biochem. Biophys. Res. Commun. 215:555-560, 1995; Yoshioka et al., Diabetologia 39: 1410-1411, 1996; Fujisawa et al., Diabetologia 39:349-352, 1996; Kurabayashi et al., Diabetes 45:1358-1363, 1996; Zhang et al., Diabetologia 39:1505-1511, 1996; Widen et al., New Engl. J. Med. 333:348-351, 1995; Clement etal., New Engl. J. Med. 333:352-354, 1995) describing the existence of a positive correlation between an Arg to Trp mutation at position 64 in the human &bgr;
3
-AR (h&bgr;
3
-AR) gene and the early onset of non-insulin dependent diabetes mellitus (NIDDM) (Walston et al, supra; Yoshioka et al., supra; Fujisawa et al., supra; Kurabayashi et al., supra; Widen et al., supra), insulin resistance (Walston et al., supra), increased weight gain (Kadowaki et al., supra; Yoshioka et al. supra; Fujisawa et al. supra; Zhang et al, supra; Clement et al., supra) and abdominal obesity (Walston et al., supra) seemed to validate the role of the &bgr;
3
-AR in the development of obesity and diabetes. However, other investigators have failed to observe any correlation between the prevalences of the Tr&bgr;
64
Arg mutation and obesity or diabetes in the patient populations they have studied (Li, et al., Diabetologia 39:857-860, 1996; Elbein et al., J. Clin. Endocrinol. Metab. 81:4422-4427, 1996; Ueda et al., Metabolism 46:199-202, 1997; Awata et al. Diabetes Care 19:271-272, 1996). Further, the pharmacology and biology of the wild type and mutated receptors did not differ when compared in cell based systems (Candelore et al., Endocrinology 137:2638-2641, 1996; Pietri-Rouxel et al., Eur. J. Biochem. 247:1174-11791 1997), although it does appear that the mutations cause less accumulation of cAMP, indicating diminished signal transduction efficiency in the cells that contained mutated h&bgr;
3
-AR. The physiological significance of this observation is not yet clear. The success achieved in the treatment of obesity and diabetes in rodent models with selective &bgr;3-AR agonists supported a role for this receptor as a therapeutic target and has prompted a great effort to develop compounds with a high affinity and selectivity towards the human &bgr;3-AR.
Despite the approximately 80% homology in amino acid sequences and similar adipose tissue expression pattern, human and mouse &bgr;
3
-ARs differ in two very important ways. First, the mouse &bgr;3-AR shows a high affinity and selectivity for certain &bgr;
3
-AR specific agonists, while the human receptor has little or no affinity for the same agonists when tested in CHO cells stably transfected with human &bgr;
3
AR (Liggett et al., Molecular Pharmacology, 42:634-637, 1992). On the other hand, agonists that show high affinity and selectivity in &bgr;
3
-AR transfected CHO cells that express a high level of human &bgr;
3
-ARs have little or no activity in vivo (when tested in non human primates and clinical trials). Their lack of robust activity may be due to pharmacokinetic or metabolic issues, but another reason for this discrepancy may be the low level of &bgr;
3
-AR expression in target tissues (Wilson et al., The Journal of Pharmacology and Experimental Therapeutics 279:214-221, 1996).
Second, in rodents &bgr;
3
-AR mRNA is predominantly detected in brown and white adipose tissue (Granneman et al., Mol. Pharmacol. 40:895-899, 1991; Muzzin et al., J. Biol Chem. 266:24053-24058, 1991), while in man &bgr;
3
-ARs are expressed in BAT but appear to have little or no expression in WAT (Krief et al., J. Clin. Invest. 91:344-349, 1993; Thomas and Liggett, Mol. Pharmacol., 43:343-343, 1991). However, other studies support the existence of functionally active &bgr;
3
-ARs in human WAT, showing an increase in lipolysis and glycerol formation after treatment with CGP-1 2177 (a selective agonist for the human &bgr;
3
-AR) both in vivo (Lonnqvist, Br. J. Pharmacol., 1993) and in vitro (Enocksson et al., J. Clin. Invest. 95:2239-2245, 1995). The significance of these studies in terms of the importance of &bgr;
3
-ARs in the regulation of WAT physiology remains to be proven, since CGP-12177 might interact with other as yet undescribed adrenergic receptors (Kaumann, A. J., Trends in Pharmacological Sciences 18:70-76, 1997).
The low affinity for synthetic agonists together with a low level of expression of h&bgr;
3
-ARs in WAT and the relatively small amount of BAT in humans may explain why agonists developed so far have been inactive in man.
Although the beneficial effects of an increased presence of &bgr;
3
-ARs in man can be envisioned, research leading to a better understanding of the factors that regulate the h&bgr;
3
-AR gene expression has been limited. Given the possibility that increased expression of &bgr;
3
-AR in humans will lead to an increased accessibility of receptors in WAT and BAT, as well as to the recruitment of more brown adipocytes, there is a need in the art to better understand mechanisms and factors that regulate transcription of h&bgr;
3
-ARs.
This problem is rendered more complex due to the lack of human brown and white adipose tissue cell lines. Although the genes for mouse (Nahmias et al., EMBO J. 10:3721-3727, 1991), rat (Granneman et al., Mol. Pharmacol. 40:895-899, 1991), and human &bgr;
3
-ARs (Granneman et al., Mol. Pharmacol. 44:264-270, 1993); Granneman et al., Mol. Pharmacol. 42:964-970, 1992; Emorine et al., Science 245:1118-1121) have been cloned, little additional data is available about the structure of regulatory regions and possible transcription factors close to the proximal promoter that may play a role in &bgr;
3
-AR transcriptional regulation (Liggett et al., J. DNA Sequencing and Mapping 2:61-63, 1991). Thus, there is a need in the art to provide the endogenous regulatory sequences of &bgr;
3
-AR.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides an isolated nucleic acid comprising a nucleotide s
Duzic Emir
Susulic Vedrana S.
Leffers Gerry
Wyeth
LandOfFree
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