Jin-Oh Kwak

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells.

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Co-localization and interaction of organic anion transporter 1 with caveolin-2 in rat kidney.

The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OATs functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1s function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.

Evidence for Na+/Ca2+ exchanger 1 association with caveolin-1 and -2 in C6 glioma cells.

The purpose of this study is to understand the interaction of Na+‐Ca2+ exchanger (NCX1), that is one of the essential regulators of Ca2+ homeostasis, with caveolin (Cav)‐1 and Cav‐2 in Cav‐3 null cell (rat C6 glioma cell). Both mRNA and protein expression of NCX1, Cav‐1 and Cav‐2 was observed, but no expression of mRNA and protein of Cav‐3 were observed in C6 glioma cells. In isolated caveolae‐enriched membrane fraction, the NCX1, Cav‐1 and Cav‐2 proteins localized in same fractions. The experiment of immuno‐precipitation showed complex formation between the NCX1 and Cavs. Confocal microscopy also supported co‐localization of NCX1 and Cavs at the plasma membrane. Functionally, sodium‐free induced forward mode of NCX1 attenuated by Cav‐1 antisense ODN. When treated cells with Cav‐2 antisense ODN, both reverse and forward mode of NCX1 was attenuated. From these results, in the Cav‐3 lacking cells, the function of NCX1 might be regulated by binding with Cavs. Considering the decrement of NCX1 activity by antisense ODNs, caveolins may play an important role in diverse of pathophysiological process of NCX1‐related disorders in the body. IUBMB Life, 56: 621‐627, 2004

Evidence for Rat Organic Anion Transporter 3 Association with Caveolin‐1 in Rat Kidney

The rat organic anion transporter 3 (rOAT3) has recently been identified as the third isoform of the OAT family. The mechanisms that regulate rOAT3s functions remain to be elucidated. rOAT3 contributes for moving a number of negatively charged organic compounds between cells and their extracellular milieu. Caveolin (Cav) also plays a role as a membrane transporter. To address the relationship of these two proteins, we investigated the protein‐protein interaction between rOAT3 and Cav‐1. The rOAT3 mRNA and protein expression were observed in the rat kidney, and the expressions of Cav‐1 mRNA and protein were also detected in the kidney. Confocal microscopy of the immuno‐cytochemistry experiments using primary cultured renal proximal tubular cells showed that rOAT3 and Cav‐1 were co‐localized at the plasma membrane. This finding was confirmed by Western blot analysis using isolated caveolae‐enriched membrane fractions from the rat kidney and immuno‐precipitation experimentation. When rOAT3s synthesized cRNA of rOAT3 along with the antisense oligo deoxynucleotide of Xenopus Cav‐1 were co‐injected into Xenopus oocytes, the [3H] estrone sulfate uptake was significantly decreased. These findings suggest that rOAT3 and caveolin‐1 share a cellular expression in the plasma membrane and Cav‐1 up‐regulates the organic anionic compound uptake via rOAT3 under normal physiological conditions. IUBMB Life, 57: 109‐117, 2005

Evidence for cyclooxygenase-1 association with caveolin-1 and -2 in cultured human embryonic kidney (HEK 293) cells

The purpose of this study was to confirm protein‐protein interaction between cyclooxygenase‐1 (COX‐1) and caveolins. The interaction of cyclooxygenase‐1 and caveolins in the cultured human embryonic kidney (HEK 293) cells was investigated using immuno‐precipitation and Western blot analysis. In HEK 293 cells, high levels of caveolin‐2 and low level of caveolin‐1 at mRNA and protein level were observed without any detectable expression of caveolin‐3. Caveolae rich membranous fractions from the HEK 293 cells contained both COX‐1 and caveolin‐1 or caveolin‐2 in same fractions. The experiments of immuno‐precipitation showed complex formation between the COX‐1 and caveolin‐1 or caveolin‐2 in the HEK 293 cells. Confocal microscopic results also support co‐localization of COX‐1 and caveolin‐1 or caveolin‐2 at the plasma membrane. Co‐localization of caveolins with cylooxygenase‐1 in caveolae suggested that caveolin would play an important role in regulating the function of COX‐1. IUBMB Life, 56: 221‐227, 2004

Identification of a novel murine organic anion transporter like protein 1 (OATLP1) expressed in the kidney

The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.

Introduction of Organic Anion Transporters (SLC22A) and a Regulatory Mechanism by Caveolins

The kidney is an important organ for controlling the volume of body fluids, electrolytic balance and excretion/reabsorption of endogenous and exogenous compounds. Among these renal functions, excretion/reabsorption of endogenous and exogenous substance is very important for the maintenance of physiological homeostasis in the body. Recently discovered organic anion transporters (OAT or SLC22A) have important roles for renal functions. It is well known as drug transporter. Several isoforms belong to SLC22A family. They showed different transport substrate spectrums and different localizations within the kidney. Their gene expressions are changed by some stimulus. The functional transport properties are regulated by protein kinase C. In addition, the function of organic anion transporters are also regulated by protein-protein interaction, such as caveolin which is compositional protein of caveolae structure. In this review, we will give an introduction of organic anion transporters and its regulatory mechanisms.