Hyi-Man Park

Small-molecule WNK inhibition regulates cardiovascular and renal function

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Gene gun-mediated in vivo analysis of tissue-specific repression of gene transcription driven by the chicken ovalbumin promoter in the liver and oviduct of laying hens.

In order to search tissue-specific elements in the 5′-upstream promoter region, gene gun was used to transfect in vivo plasmid DNAs with varying lengths of truncated ovalbumin promoter fused to the CAT reporter gene to the oviduct and liver of laying hens. The results indicated that in the oviduct, consistently high reporter gene expression was observed irrespective of the length of the truncated ovalbumin gene promoters, whereas in the liver the ovalbumin promoter extending from -3200 to +8 bp suppressed substantially the reporter gene expression compared with consistently high gene expression obtained by the ovalbumin promoters from -2800 to +8 bp or shorter length. It was concluded, therefore, that a tissue-specific silencer-like element might reside most likely in the ovalbumin gene promoter region between -3200 and -2800 bp which represses the ovalbumin gene transcription in the liver, but not in the oviduct of laying hens.

An Integrase Facilitates Long-Lasting Foreign Gene Expression In Vivo in Mouse Spermatogenic Cells

The objective of the present study was to attain long-lasting foreign gene expression in vivo in spermatogenic cells in the mouse testis for establishing spermatogenic-cell mediated gene transformation. Prior to in vivo gene transfer, surgical cryptorchidism was performed by retaining the testis into the abdominal cavity for 1 month to remove differentiated spermatogenic cells. Subsequently, in vivo gene transfer was conducted by electroporation with a lacZ reporter gene in combination with a retroviral integrase gene, and the testis was descended immediately to the scrotum to recover from the cryptorchidism, and restart spermatogenesis. At 1 month post-transfection in vivo, lacZ gene expression was detected in some spermatocyte-like cells in seminiferous tubules of the mouse testis. However, the recovery period of 1 month appeared to be too short, since no elongated and fully differentiated spermatids were found. At 2 months post-transfection, fully differentiated spermatogenic cells expressing the lacZ gene, albeit at low frequency, were detected when the integrase gene was co-transfected, while virtually no lacZ-positive cells were found in the absence of the integrase gene. It was concluded, therefore, that stable transformation of spermatogenic cells in vivo would be facilitated by integrase gene co-transfection.

Interferon regulatory factors (IRFs) repress transcription of the chicken ovalbumin gene.

Although the ovalbumin (Ov) gene has served as a model to study tissue-specific, steroid hormone-induced gene expression in vertebrates for decades, the mechanisms responsible for regulating this gene remain elusive. Ov is repressed in non-oviduct tissue and in estrogen-deprived oviduct by a strong repressor site located from -130 to -100 and designated CAR for COUP-TF adjacent repressor. The goal of this study was to identify the CAR binding protein(s). A transcription factor database search revealed that a putative interferon-stimulated response element (ISRE), which binds interferon regulatory factors (IRFs), is located in this region. Gel mobility shift assays demonstrated that the protein(s) binding to the CAR site is recognized by an IRF antibody and that mutations in the ISRE abolish that binding. In hopes of identifying the IRF(s) responsible for the tissue-specific regulation of Ov, mRNA levels for IRFs-4, -8, and -10 were measured in seven tissues from chicks treated with or without estrogen. PCR experiments showed that both IRF-8 and -10 are expressed in all chick tissues tested whereas IRF-4 has a much more limited expression pattern. Transfection experiments with OvCAT (chloramphenicol acetyltransferase) reporter constructs demonstrated that both IRF-4 and IRF-10 are capable of repressing the Ov gene even in the presence of steroid hormones and that nucleotides in the ISRE are required for repression. These experiments indicate that the repressor activity associated with the CAR site is mediated by IRF family members and suggest that IRF members also repress Ov in non-oviduct tissues.

Foreign gene expression by in vivo gene electroporation in the quail testis.

To investigate whether or not foreign gene expression is attained in the testis of living Japanese quails, a firefly luciferase reporter gene was transfected by in vivo electroporation (EP), and transcriptional activity of different promoters was compared. In addition, the effect of the Epstein-Barr virus self-replication sequence was also tested. The results showed that luciferase activity in the testis reached almost a plateau value at 50 V. Under this EP condition, no difference was found in transcriptional activity between the simian virus 40 (SV40) and miw promoters. The reporter gene expression in the quail testis was observed over 28 days after in vivo gene EP, although the activity gradually decreased, and the presence of the Epstein-Barr virus self-replication sequence in the SV40 promoter did not significantly prolong the luciferase activity. These results suggest that in vivo gene EP confers strong, though transient, foreign gene expression in the Japanese quail, and it may provide a new powerful approach for studies on transcriptional regulation of genes during proliferation and differentiation of spermatogenic cells in the quail testis.

Estrogen receptor is not primarily responsible for altered responsiveness of ovalbumin mRNA induction in the oviduct from genetically selected high- and low-albumen chicken lines.

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1, the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.

Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

Transcriptional Activities of Viral and Cellular Promoters In Vivo in the Oviduct of Living Chickens

The experiments described herein were designed to compare the transcriptional activities of promoters in vitro and in vivo in oviductal cells of chickens with or without steroid hormones. Three viral and cellular promoters fused to the chloramphenicol acetyltransferase reporter gene were transfected in vitro into primary cultured oviduct cells from estrogen-stimulated immature chicks, and in vivo into oviduct tubular gland cells from laying hens. For transferring DNA to the oviduct of laying hens, in vivo electroporation was used. The results indicated that steroid administration induced the in vivo transcriptional activity of these promoters with steroid response elements as observed with primary cultures of oviduct cells in vitro The present finding implicates that the localized in vivo gene transfer technique by electroporation would serve as a useful tool to elucidate the mechanism of steroid-induced transcription of the oviduct-specific genes in the chicken.

Human Erythropoietin Gene Expression in Primary Cultured Oviduct Cells from Estrogen-Stimulated Chicks

The present study was conducted to investigate whether or not the human erythropoietin gene is expressed in primary cultured oviduct cells from estrogen-stimulated chicks. The erythropoietin gene was transfected by calcium phosphate precipitation, and erythropoietin mRNA level was determined by RNA:RNA solution hybridization during the culture periods for up to 72hrs. The results indicated that the human erythropoietin gene was actually transcribed in the chicken oviduct cells, and erythropoietin mRNA concentration in-creased on the basis of both per cells and per total nucleic acids as the culture period went by, reaching maximal values at 72hrs.

Enhanced Transcriptional Activity by Modifying the Chicken Ovalbumin Gene Promoter in the Oviduct Cell of Chickens

The present study was conducted with chicken oviduct cells to characterize the function of the NF-1 like factor binding element (NF1BE), and the half estrogen-response-element direct repeat (1/2EREDR), both of which reside the 5′-flanking region of the chicken ovalbumin (OV) gene, and have been previously reported to act like enhancers in CV1 and HeLa cells, respectively. The chloramphenicol acetyltransferase (CAT) reporter gene fused downstream to the OV promoter of a variety of length with or without NF1BE andl/2EREDR was transfected to primary-cultured oviduct cells taken from estrogen-stimulated immature chicks. The results of CAT assays indicated that in the oviduct cells, 1/2EREDR enhanced transcriptional activity with the loss of steroid responsiveness, while the NF1BE did not, suggesting that the reported mechanisms of the 1/2EREDR in HeLa cells and of the NF1BE in CV1 cells might be different from those in the oviduct cells where these two elements should originally be functioning.