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Biomedical subjects

R W Park

Publications and source records attributed to R W Park.

At least 19 recordsLinked to original sources

Bone-protecting effect of safflower seeds in ovariectomized rats.

Safflower (Carthamus tinctorius L.) seeds have long been clinically used in Korea to promote bone formation and prevent osteoporosis. However, the beneficial effect has not been scientifically evaluated. Thus, in the present study we investigated whether phytoestrogen rich safflower seeds reduce bone loss in ovariectomized rats. Female Sprague-Dawley rats were subjected to bilateral ovariectomy or sham surgery. One week after the operation, ovariectomized rats were either fed a diet containing defatted safflower seeds or injected with 17b-estradiol (E2) for 4 weeks. As expected, ovariectomy resulted in a dramatic reduction in trabecular bone mass of the proximal tibia, increase in deposition of marrow fat, and in uterine atrophy. E2 treatment almost completely prevented bone loss as well as marrow adiposity, as examined by scanning electron microscopy and histomorphometry. Safflower seeds partially prevented ovariectomy-induced bone loss and slightly reduced marrow adiposity. Safflower seeds, in contrast to E2, exerted very weak uterotrophic action. In an attempt to elucidate the underlying mechanisms, effect of polyphenolic compounds extracted from safflower seeds on proliferation of osteoblast-like cells was also assessed in vitro. The mixed polyphenolic compounds stimulated growth of ROS 17/2.8 osteoblast-like cells in a dose-dependent manner (5-100 mg/ml), as potently as E2 and genistein. The present data provide the first direct in vivo evidence that safflower seeds have a protecting effect on bone loss caused by estrogen deficiency, without substantial effect on the uterus. The beneficial effect of safflower seeds may be mediated, at least in part, by the stimulating effect of polyphenolic compounds on proliferation of osteoblasts.

Animal Feed↗

Transcriptional repression of type I procollagen genes during adipocyte differentiation.

Adipocyte differentiation is a very complex process in which whole-cell changes are accompanied. Among them, type I procollagen gene has been shown to specifically decrease during adipocyte differentiation; however, little is known about the molecular mechanism. To examine how type I procollagen gene expression is regulated at the level of transcription during adipocyte differentiation, 3T3-L1 preadipocyte cell line was used as an in vitro model. Northern blot analysis demonstrated that mRNA expression of type I procollagen gene was dramatically reduced during adipocyte differentiation. Time-course analysis indicated that decrease in mRNA expression occurred at early stage of differentiation. Studies on several stable cell lines showed that transcriptional activities of both alpha1 and alpha2 promoters decreased significantly during adipocyte differentiation. Despite extensive deletion-promoter analyses, however, we could not identify the cis-element responsible for the switch-off of type I procollagen gene during adipocyte differentiation, suggesting that the transcriptional repression of this gene occur through general transcription machinery rather than a specific cis-element. In conclusion, down-regulation of type I procollagen mRNA expression during adipocyte differentiation is due to repression of its promoter activity through general transcription machinery.

3T3 Cells↗

The effect of early therapeutic electrical stimulation on bone mineral density in the paralyzed limbs of the rabbit.

The purpose of this animal experiment was to evaluate the changes of bone mineral density in paralyzed limbs, and to assess the effects of electrically stimulating muscle contraction upon bone mineral density (BMD) in paralyzed limbs during the four week period immediately following spinal cord injury (SCI). Ten rabbits were used for the study, spinal cords were totally transected at the T11 spine level. The paralyzed quadriceps femoris of one limb was contracted by electrical stimulation for 60-minutes daily, while the other side was not stimulated as a control. The BMD of each lower limb was measured by Dual Photon Absorptiometry before and four weeks after acute SCI. BMD of both limbs decreased in all rabbits four weeks after SCI. The decrease in BMD for stimulated and non-stimulated limbs was 6.130 +/- 3.212% and 9.098 +/- 3.831%, respectively during the four-week period after SCI. The BMD of stimulated limbs decreased significantly less than that of the non-stimulated limbs. Electrically induced muscular contraction reduced bone mineral loss in the paralyzed limb during the early stage of SCI in the rabbit.

Animals↗

Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3.

betaig-h3 is a transforming growth factor-beta-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant betaig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type betaig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for betaig-h3 is alpha(3)beta(1) integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of betaig-h3, which interact with alpha(3)beta(1) integrin to mediate HCE cell adhesion to betaig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules.

Amino Acid Sequence↗

Transactivation of fibronectin promoter by HTLV-I Tax through NF-kappaB pathway.

The facts that fibronectin (FN) mRNA is elevated in cells expressing human T cell leukemia virus type I (HTLV-I) Tax protein and that Tax is known to transactivate the cellular cAMP-response element (CRE) prompted us to examine whether Tax activates the FN promoter of which CRE is thought to play an important role. We showed that Tax transactivated the FN promoter in Jurkat cells. Deletion analyses showed that the response-element resides within the promoter region of -69 bp and that an NF-kappaB-binding site at -41 bp is involved in the Tax-activation of the FN promoter. Gel-shift assays showed that DNA-protein complexes binding to the NF-kappaB site, composed of NF-kappaB p50/p65, were induced on the NF-kappaB motif at -41 bp by Tax. Overexpression of NF-kappaB enhanced the Tax-activation of the FN promoter. Our study shows that the FN promoter is transactivated by Tax through the NF-kappaB pathway.

Fibronectins↗

Transcriptional regulation of fibronectin gene by phorbol myristate acetate in hepatoma cells: a negative role for NF-kappaB.

The transcriptional regulation of the fibronectin (FN) gene in hepatoma cells by phorbol myristate acetate (PMA) was investigated. PMA increased the synthesis and mRNA levels of FN and its promoter activity in Hep3B hepatoma cells. The PMA-induced activation of FN expression was blocked by a protein kinase C (PKC) inhibitor and did not require a new protein synthesis. Deletion analysis revealed that the sequence between positions -69 and +136 of the FN gene was responsible for the PMA induction. Two PMA-inducible nuclear protein complexes were found to bind to a putative NF-kappaB site at -41 and were identified as a p65/p50 heterodimer and a p50/50 homodimer of NF-kappaB family. Mutations in the -41 NF-kappaB site, however, did not block the PMA induction of the FN promoter but rather enhanced it. Overexpression of p65 increased the FN promoter activity. While overexpression of p50 alone did not affect the promoter activity, it decreased the p65-induced activation of the FN promoter. Mutations in the -41 NF-kappaB site attenuated the p50-mediated suppression of the p65 transactivation of the FN promoter. Deletion of the sequence between +1 and +136 decreased the basal and PMA-induced activities of the FN promoter. This study shows that PMA induces the transcription of the FN gene in hepatoma cells via the PKC pathway. The DNA sequence between +1 and +136 is responsible, at least in part, for the PMA-induced activation of the FN gene, while the -41 NF-kappaB binding site plays as a negative regulatory element for it. In addition, this study is the first to show a role for NF-kappaB p65 in the transcriptional activation of the FN gene.

Base Sequence↗

A TGF-beta-inducible cell adhesion molecule, betaig-h3, is downregulated in melorheostosis and involved in osteogenesis.

Melorheostosis is a rare bone disease characterized by linear hyperostosis and associated soft tissue abnormalities. The skin overlying the involved bone lesion is often tense, shiny, erythematous, and scleodermatous. In order to look for genes differentially expressed between the normal and involved skin, we cultured skin fibroblasts from the skin lesions of several afflicted patients, and identified differentially expressed genes by reverse dot-blot hybridization. We found that the genes human TGF-beta-induced gene product (betaig-h3), osteoblast-specific factor 2, osteonectin, fibronectin, and type I collagen were all downregulated in the affected skin fibroblasts, with betaig-h3 the most significantly affected. The expression of betaig-h3 was induced by TGF-beta in both affected and normal fibroblasts. In an effort to determine the mechanism of bone and skin abnormalities in melorheostosis, we made recombinant betaig-h3. Both immobilized and soluble recombinant betaig-h3 proteins with or without an RGD motif inhibited bone nodule formation of osteoblasts in vitro. Taken together, our results suggest that altered expression of several adhesion proteins may contribute to the development of hyperostosis and concomitant soft tissue abnormalities of melorheostosis, with betaig-h3 in particular playing an important role in osteogenesis.

Animals↗

A case of nephrogenic diabetes insipidus caused by obstructive uropathy due to prostate cancer.

Nephrogenic diabetes insipidus (DI) secondary to chronic urinary tract obstruction is a rare disease. The exact cause is unknown but it is likely that increased collecting duct pressures cause damage to the tubular epithelium, resulting in insensitivity to the action of arginine-vasopressin (AVP). A 77-year-old man complaining of polyuria and polydipsia was treated with alpha glucosidase inhibitor under the impression of polyuria due to diabetes mellitus. But his symptoms did not improve. Water deprivation and AVP administration study revealed that the patient had nephrogenic DI. Urinary tract obstruction due to an enlarged prostate was suggested as a principal cause of nephrogenic DI. The patient underwent transurethral resection of the prostate and bilateral subcapsular orchiectomy. After surgery, the urine osmolarity was normalized and the patient became symptom-free. We report a case of nephrogenic DI due to obstructive uropathy which was cured by surgery eliminating obstruction.

Adenocarcinoma↗

Induction of fibronectin gene expression by inhibitors of protein phosphatase type 2B in normal and transformed fibroblasts.

Two intracellular signal pathways mediated by cAMP and protein kinase C (PKC) were involved in the regulation of FN gene expression (Lee et al., Exp. Mol. Med. 30: 240, 1998). In this study, a possible involvement of protein phosphatase-dependent pathways in the regulation of FN gene expression was investigated by using protein phosphatase type 2B (PP2B) inhibitors, cyclosporin A and ascomycin. Both cyclosporin A and ascomycin increased the levels of FN mRNA in WI-38 human lung fibroblasts and the SV40-transformed WI-38 cells but not in MC3T3-E1 osteoblasts. The expression of FN appears to increase from six hours up to 48 hours after treatment suggesting that it is not an immediate effect. In addition, this effect required a new protein synthesis. Neither cyclosporin A nor ascomycin affects the phorbol myristate acetate (PMA)-induced stimulation of FN gene expression and the same result occurred in vice versa suggesting the mechanism of PMA and cyclosporin A/ascomycin in the regulation of FN gene expression may share a common downstream pathway. Taken together, this study suggests that PP2B is involved in the regulation of FN gene expression in normal and transformed fibroblasts but not in osteoblasts.

Animals↗

Regulation of fibronectin gene expression by cyclic AMP and phorbol myristate acetate in HT-1080 human fibrosarcoma cells.

We studied the regulation of fibronectin (FN) gene expression by cAMP and phorbol-12-myristate-13-acetate (PMA) in HT-1080 human fibrosarcoma cells. Dibutyryl cAMP increased FN synthesis and mRNA levels, while PMA inhibited the cAMP-induced FN synthesis. In transient transfection assays, cAMP increased FN promoter activity, while PMA paradoxically enhanced the cAMP-induced promoter activity. Stable transfection experiments, however, showed that neither cAMP or PMA alone nor together affected FN promoter activity. These results suggest that PMA antagonizes the cAMP-induced FN gene expression and that both the action of cAMP and the inhibition of its action by PMA may occur at the posttranscriptional level in HT-1080 cells.

Blotting, Northern↗

Factors affecting the heat resistance of Escherichia coli O157:H7.

Escherichia coli O157:H7 has been reported as being not particularly heat resistant. However, several factors which might increase its heat resistance have been investigated in this study using five strains. Increase in growth temperature to 40 degrees C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 degrees C, and variable heating rate (1 degree C min-1 to 23 degrees C min-1) had no dramatic effect on heat resistance. Growth phase had a marked impact on heat resistance; late stationary phase cells were more heat-resistant than were log phase cells. The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells. The addition of polyphosphates to the heating medium did not increase heat resistance. A reduction in water activity of the heating medium from 0.995 to levels between 0.980 and 0.960 also resulted in a marked increase in heat resistance. This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil. Survivors were detected even after a heat treatment at 60 degrees C for 1 h or 70 degrees C for 5 min. It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance.

Animals↗

Antagonistic regulation of protein kinase C-induced stimulation of fibronectin synthesis by cyclic AMP in human lung fibroblasts.

We studied the interaction between cAMP and protein kinase C (PKC) signaling pathways in the regulation of fibronectin synthesis in human lung fibroblasts. Phorbol myristate acetate (PMA), a PKC activator, stimulated fibronectin synthesis and its mRNA expression in both normal and transformed human lung fibroblasts (WI-38 and WI-38 VA13, respectively). On the other hand, dibutyryl cAMP (Bt2cAMP), a cAMP analogue, did not alter fibronectin synthesis in both cell lines. The combined treatment of Bt2cAMP with PMA, however, suppressed the PMA-induced stimulation of fibronectin synthesis and mRNA expression in these cells. This study shows that cAMP pathway antagonizes PKC pathway in regulating fibronectin synthesis in human lung fibroblasts and provides an example of antagonistic interaction between cAMP and PKC signaling pathways.

Bucladesine↗

c-Krox binds to several sites in the promoter of both mouse type I collagen genes. Structure/function study and developmental expression analysis.

We have previously shown that c-Krox is a zinc finger protein that can increases the transcriptional activity of the mouse alpha1(I) collagen promoter through its binding to two GC-rich sequences (Galéra, P., Musso, M., Ducy, P., and Karsenty, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9372-9376). In this report we show that c-Krox can bind to an additional site in the promoter of the alpha1(I) collagen gene and to three sites in the promoter of the alpha2(I) collagen gene, the other gene coding for type I collagen. One of the binding sites present in both promoters is adjacent to the CCAAT box. We have performed a structure/function analysis of c-Krox locating the transactivation domain in the zinc finger and C-terminal domains and the dimerization domain in the C-terminal end of the protein. We also demonstrate that c-Krox is an early response gene, whose expression is detectable as early as 9.5-day postcoitum in mouse embryos. Whole-mount in situ hybridization shows that c-Krox is expressed in dermatomes, the somite derivatives that generate dermis, and section in situ hybridization shows that c-Krox and alpha1(I) collagen mRNAs colocalized in skin but not in bone during development. This result is consistent with the predominant expression of c-Krox in skin in postnatal life. Thus, our findings suggest that c-Krox is one transcription factor controlling the coordinated expression of the two type I collagen genes in skin.

3T3 Cells↗

Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells.

Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4-10), bone matrix formation/maturation (days 10-16), and mineralization stages (days 16-30). During the proliferation period (days 4-10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10-16), type I collagen expression and biosynthesis, fibronectin, TGF-beta 1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-beta 1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16-30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation.

Alkaline Phosphatase↗

Okadaic acid increases fibronectin synthesis in MC3T3-E1 cells.

Okadaic acid, a potent specific inhibitor of serine/threonine protein phosphatases type 1 and 2A, affects several gene expressions in various cells. To determine whether okadaic acid affects the expression of fibronectin in MC3T3-E1 cells, we measured mRNA level and synthesis of fibronectin by Northern blot hybridization and immunoprecipitation methods, respectively. Okadaic acid (10-50 ng/ml) increased both mRNA level and synthesis of fibronectin in a dose-dependent manner. The increase of fibronectin mRNA by okadaic acid was strongly attenuated by the inhibition of new protein synthesis. The results indicate that okadaic acid, inhibitor of protein phosphatases, increases fibronectin synthesis in MC3T3-E1 cells.

Animals↗

Stimulation of fibronectin synthesis through the protein kinase C signalling pathway in normal and transformed human lung fibroblasts.

We examined the role of the protein kinase C (PKC) signaling pathway in the stimulation of fibronectin synthesis in both normal and transformed human lung fibroblasts. Phorbol myristate acetate (PMA), a potent PKC activator, stimulated fibronectin synthesis in both normal and transformed fibroblasts in a time and dose dependent fashion. Down-regulation of PKC by prior exposure of cells to a high concentration of PMA blocked the increase in fibronectin synthesis and mRNA levels induced by PMA. Bisindolylmaleimide, a specific inhibitor of PKC, also abolished the PMA-induced fibronectin synthesis. 4 alpha-phorbol didecanoate, an inactive phorbol ester, failed to affect fibronectin synthesis. These data suggest that PMA stimulates fibronectin synthesis and gene expression through the PKC signaling pathway in both normal and transformed human lung fibroblasts.

Carcinogens↗

Bisindolylmaleimide inhibits the PMA-induced down-regulation of collagen synthesis in fibroblasts.

We assessed the role of protein kinase C (PKC) in the regulation of collagen synthesis. Two PKC activators, Phorbol 12-myristate 13-acetate (PMA) and 1-oleyl 2-acetyl-sn-glycerol (OAG), decreased the relative rate of collagen synthesis in a dose- and time-dependent manner in fibroblasts, whereas an inactive phorbol ester, 4 alpha-phorbol didecanoate failed to affect the collagen synthesis. In PKC-depleted cells both PMA and OAG were unable to inhibit collagen synthesis. Bisindolylmaleimide, a specific inhibitor of PKC, completely abrogated PMA-induced inhibition of collagen synthesis in a dose-dependent fashion while two other PKC inhibitors with low specificity, H7 and staurosporin failed to block PMA effect on collagen synthesis. The results provide evidence that collagen synthesis is regulated through the signal pathway involving PKC activation.

Cells, Cultured↗

Regulation of type I collagen genes expression.

Type I collagen, the most abundant protein of the body, is preferentially synthesized in bone, dermis, and tendons by two cell types, the osteoblast and the fibroblast. The expression of type I collagen is increased in the various forms of fibrosis such as lung, liver, bone marrow fibrosis and scleroderma. Type I collagen is a heterotrimer molecule consisting of two alpha 1(I) chains and one alpha 2(I) chain. The two polypeptide chains are synthesized in a 2:1 stoichiometry. The same 2:1 ratio is observed for the rate of synthesis of the corresponding mRNAs. One hypothesis that would explain how this coregulation occurs at the transcriptional level is that common cis-acting elements are present on both genes. These common regulatory elements would bind identical transcription factors displaying the same function. The characterization of the various regulatory elements present in these genes would foster our understanding of the molecular mechanisms controlling type I collagen gene expression in normal and in pathological situations. Over the past few years, several laboratories have identified cis-acting elements in the promoters of the COL1A1 and COL1A2 genes. At least, two of these cis-acting elements are common to both promoters. One is centered by a pentanucleotide CCAAT and binds a ubiquitously expressed heteromeric CCAAT binding factor. A second one is centered by a G-rich region and it binds a new transcription factor called C-Krox.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen↗