Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts
Jie Zhang, Yifeng Deng, Huijie Ma, Jiafa Hou, and ZhenLei Zhou1
The Bone Biological Laboratory of Livestock and Poultry, College of Veterinary Medicine, Nanjing Agricultural
University, Nanjing 210095, Jiangsu Province, China
ABSTRACT Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIRENcontrol) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P < 0.01) and 27.9% (P < 0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P < 0.01) and 29.8% (P < 0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during
Key words: calbindin-D28K, chicken, osteoblasts, RNAi, transient receptor potential vanilloid 6 2015 Poultry Science 94:395–401 http://dx.doi.org/10.3382/ps/peu071
Intracellular Ca2+, as an important second messenger in cells, has been shown to regulate numerous functions, including signal transduction and differentiation. Therefore, the homeostasis of intracellular
Ca2+ is crucial for normal activity. However, little is known about the mechanisms mediating intracellular
Ca2+ signaling in osteoblasts (Blair et al., 2007).
Transient receptor potential vanilloid 6 (TRPV6), as a member of the TRP family, is a highly selective
Ca2+ channel and is considered the primary protein responsible for the absorption of transcellular
Ca2+. In vitro studies have demonstrated that a negatively charged amino acid (D) within the mouse
TRPV6 protein plays a critical role in the Ca2+
C© 2015 Poultry Science Association Inc.
Received July 7, 2014.
Accepted October 10, 2014. 1Corresponding author: firstname.lastname@example.org permeation of the channel (Bianco et al., 2007;
Nijenhuis et al., 2005; Woudenberg-Vrenken et al., 2012). In addition, some studies have offered both functional and genetic evidence that transcellular epithelial calcium uptake is vital for bone formation and the maintenance of life (Vanoevelen et al., 2011). In humans, TRPV6 is considered a crucial gate in the control of the calcium level, particularly under conditions associated with a low calcium diet when the channel is maximally upregulated (Kovacs et al., 2013).
Ca2+ transport channels include plasma membrane
Ca2+ ATPase 1b (PMCA1b), TRPV6, and Na+/Ca2+ exchangers (NCX1). The calcium-binding protein calbindin-D28K also plays a pivotal role in the transepithelial translocation of calcium molecules. According to the most accepted model of calcium transport across the epithelia, calcium enters the cell through specific epithelial calcium channels (TRPV5 or TRPV6), moves throughout the cytoplasm bound to calcium-binding proteins, such as calbindin-D28K and calbindin-D9K, and 395 at N ew
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D ow nloaded from 396 ZHANG ET AL.
Table 1. The oligonucleotide sequences of the shRNAs used in this study. shRNA Sequence (5′-3′) shRNA-TRPV6–1
Antisense aattcacgcgtAAAAAAGCAGACAGCTCTTCACATTTTCAAGAGAAATGTGAAGAGCTGTCTGCg shRNA-TRPV6–2
Antisense aattcacgcgtAAAAAAGCAGCAGTGAACCAGAATATTCAAGAGATATTCTGGTTCACTGCTGCg shRNA-TRPV6–3
Antisense aattcacgcgtAAAAAAGGGTAACACTGTTCTTCACTTCAAGAGAGTGAAGAACAGTGTTACCCg shRNA-control
Antisense aattcACGCGTAAAAAAAGTTATAGCCGTATCCACCTTCAAGAGAGGTGGATACGGCTATAACTg is extruded to the extracellular medium through the action of PMCA1b and NCX1 (Hoenderop et al., 2005).
Moreover, these proteins are expressed in human and mouse osteoblast-like cells (van der Eerden et al., 2012).
TRPV6 and PMCA1b were found to be highly expressed in preeclampsia, whereas the expression of
NCX1 was reduced in the fetus (Yang et al., 2013).
The expression levels of TRPV6 and calbindin-D9K increase in response to a low-Ca2+ diet in both wildtype and TRPV6D541A/D541A mice (WoudenbergVrenken et al., 2012). The expression levels of TRPV6 and NCX1 are upregulated in maternal and central placentas of calbindin-D28K-knockout mice but were induced in both maternal and fetal placentas in calbindinD9K-knockout mice during pregnancy (Koo et al., 2012).