Introduction Inherited monogenetic endocrine disorders are attractive candidates for gene therapy. The optimal gene therapy target is the physiological production site of the deficient hormone (e.g. pituitary gland somatotropes for
Antonis Voutetakis, Ioannis Bossis, Marc R Kok, Weitian Zhang, Jianghua Wang, Ana P Cotrim, Changyu Zheng, John A Chiorini, Lynnette K Nieman, and Bruce J Baum
Kyuyong Han, Haengseok Song, Irene Moon, Robert Augustin, Kelle Moley, Melissa Rogers, and Hyunjung Lim
spermatogenesis including spermatogonia differentiation and spermiation ( Kastner et al. 1996 , Cupp et al. 1999 ). Among RA receptors, roles for RARα and RXRβ in testis functions have been demonstrated in gene-targeted mouse models. RXRβ mutant mice have
Lovisa Lundholm, Milica Putnik, Michio Otsuki, Sandra Andersson, Claes Ohlsson, Jan-Åke Gustafsson, and Karin Dahlman-Wright
. 1996 , Nilsson et al . 2001 ). The receptors function as ligand-dependent transcription factors that bind to estrogen-response elements (EREs) or, for example, in association with fos and jun to activator protein 1 (AP-1) sites in target gene
CM Bishop, CJ McCabe, NJ Gittoes, PJ Butler, and JA Franklyn
Skeletal muscles are important target tissues for thyroid hormone action. The present study examines the influence of thyroid status on muscle growth and tissue-specific expression of thyroid receptor (TR) mRNA isoforms in a commercial strain of the domestic duck (Anas platyrhynchos). Four groups (n=5) of 1-week-old ducklings were rendered either hypothyroid by treatment with methimazole (6 mg 100 g(-1) body mass or 12 mg 100 g(-1) body mass), or hyperthyroid by treatment with methimazole (6 mg 100 g(-1) body mass) in combination with thyroid hormones (5 microg thyroxine (T(4)) and tri-iodothyronine (T(3)) 100 g(-1) body mass or 10 microg T(4) and T(3) 100 g(-1) body mass). Serum and tissue samples (cardiac, pectoralis and semimembranosus leg muscle, liver, pituitary and cerebral cortex) were collected from these four groups, and from a group of untreated controls, at 8 weeks of age. Development of duckling morphology was retarded in methimazole-treated birds compared with that in euthyroid controls, as evidenced by differences in skeletal dimensions, primary feather length, and body and muscle masses. Body mass was lower by 18%, and relative masses of cardiac and pectoralis muscles were lower by 28% and 32% respectively. Heterologous oligonucleotides for TR alpha, TR beta 0, TR beta2 and the housekeeping gene beta-actin were derived from chicken sequences. RT-PCR showed that TR alpha mRNA was expressed in all tissues but was not significantly affected by any of the experimental treatments. TR beta 0 mRNA expression was significantly lower in the leg muscles of ducklings treated with 12 mg methimazole 100 g(-1) body mass (0.109+/-0.047 TR:beta-actin ratio, P<0.05) compared with that in euthyroid controls (0.380+/-0.202), but was unaltered in the pectoralis and cardiac muscles. Expression of TR beta 0 mRNA was significantly higher in pectoralis (by 3.5-fold, P<0. 05), cardiac (by 4.2-fold, P=0.003) and leg (by 4.0-fold, P<0.001) muscles of ducklings treated with thyroid hormones compared with those in euthyroid controls (0.098+/-0.019, 0.822+/-0.297 and 0. 38+/-0.202 TR:beta-actin respectively). Only the pituitary gland expressed significant levels of TR beta 2 mRNA.
Mika Suzuki, Hiroshi Kobayashi, Yoshiko Tanaka, Naohiro Kanayama, and Toshihiko Terao
, the formation of the HCs–HA complex (the COC expansion) in mice is abolished by targeting the gene of bikunin, which is essential for biosynthesis of ITI ( Sato et al. 2001 , Zhuo et al. 2001 ). Ovarian histology ( Sato et al. 2001
K Toda, K Takeda, T Okada, S Akira, T Saibara, T Kaname, K Yamamura, S Onishi, and Y Shizuta
Aromatase P450 (CYP19) is an enzyme catalysing the conversion of androgens into oestrogens. We generated mice lacking aromatase activity (ArKO) by targeted disruption of Cyp19 and report the characteristic features of the ArKO ovaries and uteri as revealed by histological and biochemical analyses. ArKO females were totally infertile but there were as many developing follicles in their ovaries at 8 weeks of age as in wild-type ovaries. Nevertheless, no typical corpus luteum was observed in the ArKO ovaries. Electron microscopy revealed the presence of well-developed smooth endoplasmic reticulum, few lipid droplets and mitochondria with less organized tubular structures in the ArKO luteinized interstitial cells. These ultrastructural features were different from those of the wild-type interstitial cells, where there are many lipid droplets and mitochondria with well-developed tubular structures, characteristic of steroid-producing cells. When ArKO mice were supplemented with 17beta-oestradiol (E(2); 15 microg/mouse) every fourth day from 4 weeks of age for 1 month, increased numbers of follicles were observed in the ovaries as compared with those of untreated ArKO mice, although no typical corpus luteum was detectable. Ultrastructural analysis revealed the disappearance of the accumulated smooth endoplasmic reticulum in the luteinized interstitial cells after E(2 )supplementation. Transcripts of pro-apoptotic genes such as p53 and Bax genes were markedly elevated in the ArKO ovaries as compared with those of wild-type mice. Although E(2) supplementation did not cause suppression of the elevated expression of p53 and Bax mRNAs, it caused marked enhancement of expression levels of lactoferrin and progesterone receptor mRNAs in the uteri as well as increases in uterine wet weight. At 8 months of age, ArKO mice developed haemorrhages in the ovaries, in which follicles were nearly depleted, while age-matched wild-type females still had many ovarian follicles. Furthermore, macrophage-like cells were occasionally observed in the ArKO ovarian follicles. These results suggested that targeted disruption of Cyp19 caused anovulation and precocious depletion of ovarian follicles. Additionally, analysis of mice supplemented with E(2) demonstrated that E(2) apparently supports development of ovarian follicles, although it did not restore the defect in ovulation.
M C Velarde, S I Parisek, R R Eason, F A Simmen, and R C M Simmen
. In summary, the present results identify SLPI as an EGFR target gene. Given the recently described roles for SLPI in cell proliferation, tumor progression, and metastasis ( Zhang et al. 2002 a , Devoogdt et al. 2003 ), these findings suggest a
Eun Jig Lee and J Larry Jameson
the hypothalamus, and stores and secretes arginine vasopressin (AVP) and oxytocin. These highly-specialized features of pituitary cells afford a unique opportunity to develop targeted expression of therapeutic genes using cell type-specific approaches
E Lambertini, L Penolazzi, V Sollazzo, F Pezzetti, M de Mattei, L del Senno, GC Traina, and R Piva
Estrogen receptor (ER) alpha is expressed during osteoblast differentiation; however, both its functional role in bone metabolism and its involvement in osteoporotic pathogenesis caused by estrogen deficiency are not well understood. Loss of ER alpha gene expression could be one of the mechanisms leading to osteoporosis. Therefore, we investigated a possible modulation of ER alpha gene expression in a human osteoblastic cell line and in four primary osteoblast cultures by using a decoy strategy. Double stranded DNA molecules, mimicking a regulatory region of the ER alpha gene promoter (DNA-102) and acting as a 'silencer' in breast cancer cells, were introduced into osteoblasts as 'decoy' cis-elements to bind and functionally inactivate a putative negative transcription factor, and thus to induce ER alpha gene expression. We found that the DNA-102 molecule was able to specifically bind osteoblast nuclear proteins. Before decoy treatment, absence or variable low levels of ER alpha RNAs in the different cultures were detected. When the cells were transfected with the DNA-102 decoy, an increase in expression of ER alpha and osteoblastic markers, such as osteopontin, was observed, indicating a more differentiated osteoblastic phenotype both in the cell line and in primary cultures. These results showed that the DNA-102 sequence competes with endogenous specific negative transcription factors that may be critical for a decrease in or lack of ER alpha gene transcription. Therefore, osteoblastic transfection with the DNA-102 decoy molecule may be considered a tempting model in a putative therapeutic approach for those pathologies, such as osteoporosis, in which the decrease or loss of ER alpha expression plays a critical role in bone function.
Joyce Emons, Bas E Dutilh, Eva Decker, Heide Pirzer, Carsten Sticht, Norbert Gretz, Gudrun Rappold, Ewan R Cameron, James C Neil, Gary S Stein, Andre J van Wijnen, Jan Maarten Wit, Janine N Post, and Marcel Karperien
compared the predicted transcription factor binding with transcription factor binding data from ChIP assays and RNA interference experiments to verify the interaction of transcription factors with the regulatory region of these target genes. Materials and