The Calcitonin and Glucocorticoids Combination: Mechanistic Insights into Their Class-Effect Synergy in Experimental Arthritis

PMCID: PMC3564948This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Glucocorticoid modulation of casein gene transcription in mouse mammary gland

The influence of cortisol and prolactin on casein gene expression in the mammary gland of lactating BALB/c mice was measured by using a specific cDNA probe to 15S casein mRNA (cDNAcsn). Casein mRNA (mRNAcsn) level in the mammary gland was decreased by 85% 5 days after adrenal ablation, but then was increased 4.4-fold 12 hr after a single injection of hydrocortisone-21-acetate. An 80% decrease in serum prolactin level, induced by the prolactin inhibitor 2- bromo-α-ergocryptin (CB-154), did not alter the level of mRNAcsn in the gland. Specific transcription of the casein gene in nuclei isolated from lactating mammary glands was measured by cDNAcsn hybridization to the in vitro synthesized Hg-CTP-containing RNA (Hg-RNA), which was purified by SH-agarose chromatography. The level of the mRNAcn in Hg-RNA synthesized in the isolated nuclei was 0.09% and this was decreased 85% by α-amanitin, indicating that the mRNAcsn sequences in the Hg-RNA were the products of RNA polymerase II-directed DNA-dependent RNA synthesis. Transcription of the mRNAcsn in isolated nuclei was decreased by 70% 5 days after adrenalectomy and a single injection of the glucocorticoid then increased the transcription level 2-fold at 6 hr. Essentially no alteration of the level of transcription was detectable in mammary nuclei isolated from lactating mice with 80% decreased serum prolactin level, induced by CB-154 treatment. The results thus demonstrate a glucocorticoid involvement on the modulation of casein gene expression at the transcriptional level of control

Cloning of mouse [beta]-casein gene sequences

Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAscn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherchia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Souther transfer and hybridization to 9[32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn [alpha], [beta] and [gamma]) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn [beta].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24211/1/0000470.pd

Additive Effects of Mechanical Marrow Ablation and PTH Treatment on de Novo Bone Formation in Mature Adult Rats

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The combination elcatonin/Dexamethasone elicits a synergistic attenuation of MMP-2, TRAP-5b and CXCL5 expression.

<p>Rats were treated with collagen on Day 0 and then, from Day 11, received daily i.p. injections of elcatonin (eCT; 1.0 µg/kg) alone or together with a sub-therapeutic dose of Dexamethasone (D; 7.5 µg/kg) (Co-Tx, combination therapy). A positive control group of rats was treated with Dexamethasone (30 µg/kg). In all cases hind paw tissue extracts and plasma samples were taken at day 18. Protein levels of (A) metalloproteinase II (MMP-2) in tissue extracts, (B) plasma tartrate-resistant acid phosphatase (TRAP-5b), (C) tissue extract and (D) plasma CXCL5 were determined as described in Methods. Data are mean ± SEM of 10 rats per group. Statistical analyses by one-way ANOVA (Kruskal-Wallis test with Dunn’s post-test); *p<0.05, **p<0.01, and ***p<0.001 as compared to vehicle-treated group.</p

Elcatonin co-administration does not augment Dexamethasone-induced changes in gluconeogenesis-related liver enzyme mRNA.

<p>Quantitative real time-PCR was performed in liver tissues harvested from the hyperglycaemia experiment (see Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054299#pone-0054299-g004" target="_blank">Figure 4</a> for protocol), in which fasted rats received single dosing of Dexamethasone (µg/kg) with or without elcatonin (eCT; 1.0 µg/kg). The expression of the following genes was studied: (A) tyrosine aminotransferase (Tat); (B) phosphoenolpyruvate carboxykinase (Pck2); (C) glucose-6-phosphatase (G6pc3); (D) fructose-1,6-bisphosphase (Fbp2). Data was expressed as 2^−ΔΔCt using Gapdh as endogenous control. Values report mean ± SEM of 4 distinct rat samples. Statistical analyses by one-way ANOVA (Kruskal-Wallis test with Dunn’s post-test); *p<0.05, ns p>0.05 as compared to vehicle-treated group.</p

Elcatonin does not augment Dexamethasone-induced hyperglycaemia and ACTH suppression.

<p>Fasted rats were given a single dose of Dexamethasone (15 or 100 µg/kg i.p.) with or without 1.0 µg/kg elcatonin (eCT). Blood was collected by venepuncture and glucose was quantified: i) immediately prior to fasting, ii) prior to drug treatment, and iii) 5 h after drug treatment. Overnight fasting caused a fall in mean blood glucose from 6.19±0.10 to 4.80±0.13 mM. Blood glucose in the vehicle-treated group continued to drop, reaching 3.86±0.22 mM at the 5-hour time point. (A) Blood glucose data as measured 5 h post-treatment, and normalised for differing pre-fasting levels. (B) ACTH was assayed by EIA in blood collected by terminal cardiac puncture at 5 h post-treatment. ACTH suppression is expressed in relation to the levels quantified by ELISA in vehicle-treated animals (1.57±0.09 ng/ml). In both panels, data are mean ± SEM of 8 rats per group. Statistical analyses by one-way ANOVA (Kruskal-Wallis test with Dunn’s post-test); *p<0.05, **p<0.01, and ***p<0.001 as compared to vehicle-treated group, or by Mann-Whitney test; ^†p>0.05 in the indicated comparison.</p

Combination of elcatonin and sub-therapeutic Dexamethasone preserves articular integrity.

<p>Day 18 CIA rat hind paws from rats treated with collagen at Day 0. Elcatonin (eCT) and Dexamethasone (Dex) were given i.p. daily from Day 11, as detailed in Legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054299#pone-0054299-g001" target="_blank">Figure 1</a>. Paws were fixed, de-calcified, paraffin-embedded, sectioned and stained with haematoxylin-eosin. Light micrographs (representative of 4 animals), showing synovitis, cellular infiltrate, bone erosion and articular integrity. (A) eCT 1.0 µg/kg alone, (B) Dex 7.5 µg/kg alone, (C) co-treatment: eCT 1.0 µg/kg+Dex 7.5 µg/kg, (D) Dex 30 µg/kg. Scale bar, 300 µm.</p