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Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

ABSTRACT: Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice). iER-VCAM1 mice did not have a lethal phenotype, in contrast to the constitutive knockout of VCAM1, but adult mice exhibited physiological effects in the form of aberrant distribution of immature B-cells in blood and bone marrow. The capability to regulate knock-down strength may spark a new approach for the functional study of membrane and plasma proteins, which may especially be valuable for generating mouse models that more closely resemble disease states than classic knockouts do.

SUBMITTER: Marschall AL

PROVIDER: S-EPMC4622715 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

Marschall Andrea L J AL Single Frank N FN Schlarmann Katrin K Bosio Andreas A Strebe Nina N van den Heuvel Joop J Frenzel André A Dübel Stefan S

mAbs 20140101 6

Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice). iER-VCAM1 mice did not have a lethal phenotype, in contrast to the constitutive knockout of VCAM1, ...[more]

PMID: 25484057

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Project description:BackgroundHistidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca(2+) cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca(2+) homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca(2+) cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.Methodology/principal findingsAAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca(2+) cycling and increased activities of RyR2 and SERCA2 without change in SR Ca(2+) load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.Conclusions/significanceIncreased Ca(2+) leak and cytosolic Ca(2+) concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca(2+) cycling.

Dataset Information

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

Publications

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

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