Cultured rat calvarial osteoblast-like cells are provided with orexin type 1 receptors

Ziółkowska, Agnieszka; Ruciński, Marcin; Tortorella, Cinzia; Tyczewska, Marianna; Nussdorfer, Gastone G.; Malendowicz, Ludwik K.

doi:10.3892/ijmm.20.6.779

Cited by 9 publications

(9 citation statements)

References 36 publications

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“…The applied methods were described in previous studies (21)(22)(23)(24)(25). Total RNA was extracted from the regenerated tissue with the use of TRI Reagent (Sigma-Aldrich) and then, purified on columns (Rneasy Mini Kit; Qiagen, Hilden, Germany), as previously described (23,(25)(26)(27). The amount of total mRNA was determined by optical density at 260 nm and its purity was estimated by a 260/280 nm absorption ratio (>1.8) on a NanodDrop spectrophotometer; Thermo Scientific, Walthman, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

Tyczewska

Ruciński

Ziółkowska

et al. 2013

International Journal of Molecular Medicine

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The enucleation-induced (EI) rapid proliferation of adrenocortical cells is followed by their differentiation, the degree of which may be characterized by the expression of genes directly and indirectly involved in steroid hormone biosynthesis. In this study, out of 30,000 transcripts of genes identified by means of Affymetrix Rat Gene 1.1 ST Array, we aimed to select genes (either up- or downregulated) involved in steroidogenesis in the course of enucleation-induced adrenal regeneration. On day 1, we found 32 genes with altered expression levels, 15 were upregulated and 17 were downregulated [i.e., 3β-hydroxysteroid dehydrogenase (Hsd3β), nuclear receptor subfamily 0, group B, member 1 (Nr0b1), cytochrome P450 aldosterone synthase (Cyp11b2) and sterol O-acyltransferase 1 (Soat1)]. On day 15, the expression of only 2 genes was increased and that of 3 was decreased. The investigated genes were clustered according to an hierarchical clustering algorithm and 4 clusters were obtained. Quantitative PCR (qPCR) confirmed the much lower mRNA expression levels of steroidogenic acute regulatory protein (StAR) during the regeneration process compared to the control, while the cholesterol side-chain cleavage enzyme (cholesterol desmolase; Cyp11a1) and Hsd3β genes presented similar expression profiles throughout the entire regeneration process. Cyp11b2 mRNA levels remained very low during the whole regeneration period. Fatty acid binding protein 6 (Fabp6) was markedly upregulated, whereas hormone-sensitive lipase (Lipe) was downregulated. The expression of Soat1 was lowest on regeneration day 1 and, subsequently, its expression increased from there on, reaching levels higher than the control. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Dax-1) mRNA levels were lowest on day 1 of the experiment; however, throughout the entire experimental period, there were no statistically significant differences observed. After the initial decrease in steroidogenic factor 1 (Sf-1) mRNA levels observed on the 1st day of the experiment, a marked upregulation in its expression was observed from there on. Data from the current study strongly suggest the role of Fabp6, Lipe and Soat1 in supplying substrates of regenerating adrenocortical cells for steroid synthesis. Our results indicate that during the first days of adrenal regeneration, intense synthesis of cholesterol may occur, which is then followed by its conversion into cholesteryl esters. Moreover, our data demonstrated that in enucleation-induced regeneration, the restoration of genes involved in glucocorticoid synthesis is notably shorter than that of those involved in aldosterone synthesis.

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Section: Methodsmentioning

confidence: 99%

Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

Tyczewska

Ruciński

Ziółkowska

et al. 2013

International Journal of Molecular Medicine

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“…QPCR methods applied were described previously (20,(31)(32)(33)(34)(35)(36). The obtained cDNA was used as a template in QPCR.…”

Section: Validation Of Results By Qpcrmentioning

confidence: 99%

Cerebellin and des-cerebellin exert ACTH-like effects on corticosterone secretion and the intracellular signaling pathway gene expression in cultured rat adrenocortical cells - DNA microarray and QPCR studies

Ruciński

Ziółkowska²,

Szyszka³

et al. 2009

Int J Mol Med

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Abstract. Precerebellins (Cbln) belong to the C1q/TNF superfamily of secreted proteins which have diverse functions. They are abundantly expressed in the cerebellum, however, three of them are also expressed in the rat adrenal gland. All members of the Cbln family form homomeric and heteromeric complexes with each other in vitro and it was suggested that such complexes play a crucial role in normal development of the cerebellum. The aim of our study was to investigate whether Cbln1-derived peptides, cerebellin (CER) and des-Ser 1 -cerebellin (desCER) are involved in regulating biological functions of rat adrenocortical cells. In the primary culture of rat adrenocortical cells, 24 h exposure to CER or desCER notably stimulated corticosterone output and inhibited proliferative activity and similar effects were evoked by ACTH. To study gene transcript regulation by CER, desCER and ACTH, we applied Oligo GEArray ® DNA Microarray: Rat Signal Transduction Pathway Finder™. In relation to the control culture, 13 of the 113 transcripts present on the array were differentially expressed. These transcripts were either up-or down-regulated by ACTH and/or CER or desCER treatment. Validation of DNA Microarray data by QPCR revealed that only 5 of 13 genes studied were differentially expressed. Of those genes, Fos and Icam1 were up-regulated and Egr1 was down-regulated by ACTH, CER and desCER. The remaining two genes, Fasn (insulin signaling pathway) and Hspb1 (HSP27) (stress signaling pathway), were regulated only by CER and desCER, but not by ACTH. Thus, both CER and desCER have effects similar to and different from corticotrophin on the intracellular signaling pathway gene expression in cultured rat adrenocortical cells.

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“…Human prostate culture cells were washed two times using PBS, then the total RNA was extracted using TRI Reagent (Sigma-Aldrich, Poznan, Poland). Total RNA was purified on columns (NucleoSpin Total RNA Isolation, Qiagen GmbH, Hilden, Germany) [19][20][21][22][23]. The amount of total RNA was determined by optical density at 260 nm and its purity was estimated by 260/280 nm absorption ratio (higher than 1.8) (NanoDrop spectrophotometer, Thermo Scientific, Waltham, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

Lack of expression of preproorexin and orexin receptors genes in human normal and prostate cancer cell lines

Szyszka

Paschke

Tyczewska

et al. 2016

Folia Histochem Cytobiol.

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Introduction. Studies on expression of orexins (OXs) and their receptors in human prostate gland and human prostatic cell lines are scanty and their results contradictory. Regarding this, we carefully reinvestigated this problem on human prostatic cell lines. Material and methods. Expression of preproorexin (ppOX) (6 primer pairs), and orexin receptors 1 and 2 (OXR1, OXR2) (4 and 2 primer pairs, respectively) was assessed by conventional PCR and QPCR in human normal (PrEC, PrSc, PrSmC) and prostate carcinoma (Du145, LNCaP, and PC3) cell lines. We designed intron spanning primers and also we applied primers from earlier publications and commercially available ones. Results. With the designed primer pairs, in all studied cell lines we failed to demonstrate expression of ppOX, OXR1 and OXR2 genes at the mRNA level, while reaction products were observed in control tissues (human placenta and adrenals). Primers applied in earlier studies did not form amplification products specific for preproorexin or orexin 1 receptor. Some commercially available primers for orexin receptor 1 produced false positive results. Conclusions. We found no evidence for the presence of preproorexin-orexin receptors system genes' mRNAs in human prostate cell lines. The reported premises for these genes' expression in prostate and prostatic cell lines may have arisen either from the presence of non-prostate cells included in the samples or from faulty PCR settings.

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Cultured rat calvarial osteoblast-like cells are provided with orexin type 1 receptors

Cited by 9 publications

References 36 publications

Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

Cerebellin and des-cerebellin exert ACTH-like effects on corticosterone secretion and the intracellular signaling pathway gene expression in cultured rat adrenocortical cells - DNA microarray and QPCR studies

Lack of expression of preproorexin and orexin receptors genes in human normal and prostate cancer cell lines

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