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Differential cellular expression of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in normal human tissues: A study using high density human tissue microarrays
ARTICLE IN PRESS Annals of Anatomy 192 (2010) 7–16
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Differential cellular expression of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in normal human tissues: A study using high density human tissue microarrays Rachel V. Floyd a, Susan Wray a, Pablo Martı´n-Vasallo b, Ali Mobasheri c,n a
Physiological Laboratory, Department of Physiology, School of Biomedical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 3BX, UK Laboratorio de Biologı´a del Desarrollo, Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain c Musculoskeletal Research Group, Division of Veterinary Medicine, School of Veterinary Medicine and Science, Institute of Clinical Research and the Faculty of Medicine and Health Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK b
a r t i c l e in fo
abstract
Article history: Received 13 April 2009 Received in revised form 3 August 2009 Accepted 15 September 2009
FXYD proteins have been proposed to function as regulators of Na, K-ATPase function by lowering affinities of the system for potassium and sodium. However, their distribution in normal human tissues has not been studied. We have therefore used immunohistochemistry and semi-quantitative histomorphometric analysis to determine the relative expression at the protein level and distribution of FXYD1 (phospholemman) and FXYD2 (g subunit of Na, K-ATPase) in human Tissue MicroArrays (TMAs). Expression of FXYD1 was abundant in heart, kidney, placenta, skeletal muscle, gastric and anal mucosa, small intestine and colon. Lower FXYD1 expression was detected in uterine, intestinal and bladder smooth muscle, choroid plexus, liver, gallbladder, spleen, breast, prostate and epididymis. The tissue distribution of FXYD2 was less extensive compared to that of FXYD1. There was an abundant expression in kidney and choroid plexus and moderate expression in placenta, amniotic membranes, breast epithelium, salivary glands, pancreas and uterine endometrium. Weaker FXYD2 expression was detected in the adrenal medulla, liver, gallbladder, bladder and pancreas. The common denominator in the distribution of FXYD1 and FXYD2 was expression in highly active transport epithelia of the kidney, choroid plexus, placenta and salivary glands. This study reveals, in human tissues, the specific expression of FXYD proteins, which may associate with Na, K-ATPase in selected cell types and modulate its catalytic properties. & 2009 Elsevier GmbH. All rights reserved.
Keywords: FXYD1 (phospholemman) FXYD2 Tissue microarray Immunohisto chemistry Histomorphometric analysis
1. Introduction It is becoming increasingly recognized that small single-span membrane proteins (30 + amino acids) play important physiological roles by regulating the activity of larger macromolecular complexes that form ion permeant channels, transporters and ATPase pumps. For example, phospholamban regulates the properties of SERCA2a, the Ca-ATPase of cardiac sarcoplasmic reticulum, and its homolog sarcolipin regulates the SERCA1a CaATPase isoform of fast-twitch skeletal muscle (Odermatt et al., 1998; Simmerman and Jones, 1998). The FXYD gene family of ion transport regulators contains seven small single-span membrane proteins that share a 35-amino acid signature sequence domain, beginning with the sequence PFXYD and containing 7 invariant and 6 highly conserved amino acids (Sweadner and Rael, 2000) (see Table 1). The FXYD proteins have been proposed to function
n
Corresponding author. Tel.: + 44 115 951 6449; fax: + 44 115 951 6440. E-mail address: [email protected] (A. Mobasheri).
0940-9602/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2009.09.003
as novel tissue- and isoform-specific regulators of the plasma membrane Na, K-ATPase (Geering et al., 2003). Four of the FXYD proteins (FXYD1, FXYD2, FXYD4, FXYD7) have been shown to alter the activity of the Na, K-ATPase (Cornelius et al., 2001; Sweadner et al., 2003). Thus, the tissue- and isozyme-specific interaction of Na, K-ATPase with FXYD proteins may contribute to the proper handling of Na + and K + by Na, K-ATPase in physiological processes such as renal Na + -re-absorption, muscle contraction and neuronal excitability (Crambert and Geering, 2003). The protein products of the FXYD family have been predicted to exhibit ion channel or ion transport activity and localize in the plasma membrane. Messenger RNA transcripts encoding proteins of the FXYD family are abundant and widespread in fetal and adult mammalian tissues, particularly in epithelial tissues that perform bulk fluid and electrolyte transport including kidney, pancreas, colon, mammary glands, prostate, liver, lung and placenta. FXYD members are also abundantly expressed in cardiovascular and electrically excitable tissues (central and peripheral nervous system and various types of muscle). The best studied
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Table 1 Summary of the expression levels and cellular distribution of FXYD1 and FXYD2 in tissues represented on the CHTN2002N1 tissue microarray (http://faculty.virginia.edu/ chtn-tma/2002N1/CHTN2002normal%20TMA.html). System
Tissue
FXYD1
FXYD2
Cellular distribution
Staining intensity
Cellular distribution
Staining intensity
Cardiovascular system
aorta, smooth muscle heart, myocardium lymphatic endothelium small muscular artery (lung) small vein (intestine)
+++ ++
0 2 0 0 1
+++
0 1 0 0 0
Gastrointestinal system
esophagus, squamous mucosa gastric mucosa, antral gastric mucosa, oxyntic small intestine, mucosa colon, mucosa anus, mucosa
mucosa++ ++ + + +
1 1 2 1 1 0
++ ++ ++ ++
0 1 2 1 1 0
Genital system (male)
seminiferous tubules epididymis seminal vessicle prostate
+ +
0 1 1 0
+ ++
0 1 1 0
Hepatic (pancreatobiliary)
gallbladder liver pancreas
+++ +++ +
2 1 1
+++ ++
1 0 1
Oral, salivary & nasal
salivary gland (parotid) tonsil, squamous epithelium
++
1 0
+
2 0
Skin
skin, squamous epithelium subepidermal tissue
0 0
0 0
Breast
breast, epithelium
++
1
++
2
Genital system (female)
ectocervix endocervix endometrium, secretory fallopian tube
+++
0 0 1 0
+ +++
0 1 2 0
Endocrine system
adrenal gland, cortex adrenal gland, medulla parathyroid adenoma pituitary, anterior pituitary, posterior thyroid
+++
1 0 0 0 0 0
+ +
1 1 0 0 0 0
Soft tissue
adipose tissue, breast
0
0
Cartilage
cartilage, articular
+
1
0
Soft tissue
cartilage, bronchial
0
0
Soft tissue (muscular)
skeletal muscle smooth muscle, intestine smooth muscle, uterus
+++ ++
1 0 1
++
0 0 1
Synovium
synovium
0
0
Genital (female)
ovary, ovary, ovary, ovary,
+++ +++ +++ +++
1 1 1 1
++ +++ ++ ++
1 2 1 1
Placenta
amniotic membrane placenta, villi
+++ +++
1 2
+++ ++
2 1
Peripheral nervous system
autonomic ganglia & nerves, intestinal peripheral nerve
0
0
0
0
Central nervous system
cerebral cortex cerebellar cortex, purkinje/ granular layer choroid plexus ependymal cells meninges motor neurons (spinal cord) white matter (subcortical) hippocampus
+ +
1 1
0 0
+++ + + +
2 1 0 1 0 1
+
2 0 0 0 0 0
0 0
0 0
Lymphoid system
primary oocytes corpus luteum epithelium stroma
lymph node mucosa associated lymphoid tissue, appendix
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Table 1 (continued ) System
Tissue
FXYD1
FXYD2
Cellular distribution
Staining intensity
Cellular distribution
Staining intensity
spleen thymus tonsil
0 0 0
0 0 0
Respiratory system
alveoli bronchus, epithelium
0 0
0 0
Urological system
kidney, cortex kidney, medulla bladder, transitional epithelium
++ ++ +++
2 2 2
++ ++ +++
2 2 2
Key
Key
+ ++
No expression Scattered cells Groups of cells
+++
Most cells
0 1 2
No expression Low expression Abundant expression
The cellular distribution was assessed using a four point scale: , no expression; + , scattered cells; ++, groups of cells; +++, most cells expressed FXYD1 and FXYD2 proteins. The intensity of FXYD1 and FXYD2 immunostaining was evaluated by means of a three point scale: 0, no expression; 1, low expression; 2, abundant expression.
members of this gene family are FXYD1 and FXYD2. FXYD1 also known as phospholemman is a plasma membrane substrate for several kinases, including protein kinase A, protein kinase C, NIMA (Never in mitosis, gene A) kinase, and myotonic dystrophy kinase (Palmer et al., 1991; Moorman et al., 1992; Lu et al., 1994). It is thought to form an ion channel or regulate ion channel activity. FXYD2 is the g subunit of Na, K-ATPase (also known as HOMG2, ATP1G1 or MGC12372) and assembles with the a and b subunits of the Na, K-ATPase and forms an association tight enough to survive detergent extraction and extensive proteolysis (Or et al., 1996; Therien et al., 2001). FXYD7 is known to be expressed in the central nervous system and has been suggested to be a brainspecific regulator of Na, K-ATPase (Crambert and Geering, 2003; Crambert et al., 2003; Geering et al., 2003). Other FXYD family members are less well studied particularly FXYD3 or MAT8 (mammary tumor marker) (Morrison et al., 1995) FXYD4 or CHIF (channel-inducing factor) (Attali et al., 1995), FXYD5 or RIC (related to ion channel) (Fu and Kamps (1997)) and the relatively uncharacterized FXYD6. Although FXYD1-5 have all been shown to induce ion channel activity in experimental expression systems (Attali et al., 1995; Morrison et al., 1995; Kowdley et al., 1997; Minor et al., 1998) their distribution in normal rodent or human tissues has not been studied using high throughput immunohistochemistry. The recent proliferation of Tissue MicroArray (TMA) technology allows screening of large numbers of normal and tumor tissue specimens for gene and protein expression information and for discovering novel diagnostic and prognostic correlations. We have taken advantage of TMA technology to test the hypothesis that the distribution of FXYD1 and FXYD2 proteins in the human body is more widespread than the cardiovascular and renal systems. Nerve and muscle cells require low Na + affinity Na, K-ATPase pumps capable of rapid and efficient extrusion of intracellular Na + during periods of intense ion transport activity. The same principle may be applied to transport epithelia responsible for bulk Na + and K + transport during fundamental activities such as secretion and absorption. Therefore, in these cells/tissues co-expression of small ion transport regulators (i.e. FXYD proteins) along with the a and b subunits of Na, K-ATPase may provide the transport system with a kinetic advantage by allowing the pump to increase its rate in response to sudden and rapid rises in intracellular Na + . To test this idea we used immunohistochemistry and semi-quantitative histomorphometric
analysis to study the distribution and relative abundance of the FXYD1 and FXYD2 proteins in high density human TMAs.
2. Materials and methods 2.1. Chemicals All chemicals used were molecular biology grade and purchased from Sigma/Aldrich and Sigma Biosciences (Poole, Dorset, UK). Materials for immunohistochemistry were purchased from Vector Laboratories (Peterborough, UK) and DakoCytomation (Ely, Cambridgeshire, UK). Antibodies Polyclonal PLM-antibody, produced against a peptide CRSSIRRLSTRRR of the C terminus of rat PLM recognizes FXYD1 (Crambert et al., 2002). Polyclonal gC33 antiserum was raised against the 10-residue C terminus of the g variant (Kuster et al., 2000). The antibodies were diluted 1:300 before use. Both antisera were kind gifts of Dr. S.J.D. Karlish (Weizmann Institute of Science, Rehovoth, Israel). 2.2. High density human Tissue MicroArrays (TMAs) Tissue MicroArrays (TMAs) of formalin fixed paraffin embedded human tissues (Fig. 1) were obtained from the Cooperative Human Tissue Network (CHTN) of The National Cancer Institute (NCI), the National Institutes of Health, Bethesda, MD, USA (http://faculty.virginia.edu/chtn-tma/home.html). Medical ethics committee approval was not required for these studies. The TMAs contained 66 anonymized samples of non-neoplastic adult tissues obtained from surgical resection specimens, fixed within one hour of surgical removal from the donors. The central nervous system tissues on the TMAs were obtained from autopsy specimens within 36 hours of death. The tissues represented on the TMAs included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary and nasal, mammary, endocrine, genital tract, central and peripheral nervous system, urinary tract, skin, cartilage and synovium. In view of the extreme
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CHTN 2002N1 TMA
Microarray: X Y mm
0 0.0
CHTN2002N1
1 0.7
2 1.4
3 2.1
4 2.8
5 3.5
6 4.2
7 4.9
8 5.6
9 6.3
10 7.0
11 7.7
12 8.4
13 9.1
14 9.8
15 10.5
16 11.2
17 11.9
18 12.6
19 13.3
20 14.0
21 14.7
22 15.4
23 16.1
24 16.8
20 14.0
25 17.5 mm 14.0
19 13.3
13.3
PLACENTA
18 12.6
HEPATIC & PANCREATOBILIARY
CARDIOVASCULAR
17 11.9
ENDOCRINE
PERIPHERAL NERVOUS SYSTEM
16 11.2
LYMPHOID
12.6 11.9 11.2
15 10.5
10.5 CARDIOVASCULAR
14 9.8
HEPATIC & PANCREATOBILIARY
13 9.1 GASTROINTESTINAL
12 8.4
SOFT TISSUE
ORAL, SALIVARY
CARTILAGE
& NASAL
SOFT TISSUE
11 7.7
LYMPHOID
ENDOCRINE CENTRAL NERVOUS SYSTEM
9.8 9.1
RESPIRATORY
8.4 7.7
10 7.0
7.0
9
6.3
8
5.6
7
4.9
6
4.2
5
3.5
3.5
4
2.8
2.8
3
2.1
2
1.4
1
0.7
0
0.0 mm
6.3 GASTROINTESTINAL
GENITAL, MALE
SKIN
SOFT TISSUE
BREAST
SYNOVIUM
GENITAL, FEMALE
CENTRAL NERVOUS SYSTE
MUROLOGICAL
5.6 4.9 4.2
GENITAL, FEMALE
2.1 1.4 0.7
0.0
0.7
1.4
2.1
2.8
3.5
4.2
4.9
5.6
6.3
7.0
7.7
8.4
9.1
9.8
0.0 10.5 11.2 11.9 12.6 13.3 14.0 14.7 15.4 16.1 16.8 17.5 mm
Fig. 1. Representative Tissue MicroArray (TMA) slide consisting of 0.6 mm diameter spots of 66 normal tissue types present in the human body. The slides used throughout this study were codenamed CHTN2002N1 and obtained from the Cooperative Human Tissue Network of The National Cancer Institute, the National Institutes of Health. Higher magnification views in panel B illustrate (clockwise) hematoxylin and eosin stained spots of kidney medulla, the mucosa of the small intestine, skeletal muscle and parotid salivary gland. Solid bars represent 100 mm. The tissues cores represented on the microarray are classified by the system from which they originate (C) and are grouped together in distinct square blocks. Each cluster of spots represents tissues from a particular system (i.e. cardiovascular, urinary, etc.). For a complete list of tissues refer to http://faculty.virginia.edu/chtn-tma/2002N1/CHTN2002N1.htm.
cellular heterogeneity of complex tissues such as the brain and the wide diversity of endocrine glands, it must be pointed out that these TMAs are not a complete representation of the human body. Further details and layout of the CHTN normal human tissue TMA may be found on the CHTN website: http://faculty.virginia.edu/ chtn-tma/2002N1/CHTN2002N1X.xls.
2.3. Immunohistochemical protocol The TMAs were heated at 60 1C for 15 min to improve tissue adhesion to the charged glass slides (Fisher Plus). Prior to immunostaining, TMA slides were deparaffinized in xylene for 20 min to remove embedding media and washed in absolute ethanol for 3 min. The TMAs were gradually rehydrated in a series of alcohol baths (96%, 85% and 50%) and placed in distilled water for 5 min. Endogenous peroxidase was blocked for 1 hour in a 97% methanol solution containing 3% hydrogen peroxide and 0.01% sodium azide. Following thrice washing in phosphate buffered saline (PBS) the TMAs were subjected to antigen retrieval by incubating with 0.5% sodium dodecyl sulfate (SDS) in PBS for
10 min (Brown et al., 1996). Excess SDS was removed by incubating the TMAs for 5 min in PBS. To prevent non-specific antibody binding the TMAs were incubated for one hour at room temperature (RT) with 20% normal goat serum (NGS) in PBS containing 1% bovine serum albumin and 0.01% sodium azide. Slides were incubated overnight at 4 1C with antisera. After 24 hrs at 4 1C the slides were washed 3 times for 5 min in PBS before incubating with horseradish peroxidase labeled polymer conjugated to affinity purified goat anti-rabbit and goat anti-mouse immunoglobulins (DAKO code no. K4010) for 30 minutes (RT). The sections were washed 3 times for 5 min in PBS before applying liquid DAB+ Chromogen (DAKO; 3,30 -diaminobenzidine chromogen solution) for up to 30 s. The development of the brown colored reaction was stopped by rinsing in distilled water. The stained slides were immersed for 5 min in a bath of aqueous hematoxylin (DakoCytomation, code no. S3309) to counterstain cell nuclei. Finally the slides were washed for 5 min in running water and dehydrated in a series of graded ethanol baths before rinsing in 3 xylene baths and mounting in DPX (BDH laboratories). In some experiments (see immunohistochemical data shown in Fig. 4) the slides were incubated with alkaline phosphatase
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labeled polymer conjugated to affinity purified goat anti-rabbit and goat anti-mouse immunoglobulins instead of horseradish peroxidase labeled polymer. In these experiments the Fast Red Substrate System (tablets containing naphthol phosphate, fast red and levamisole) was used as a chromogen (DakoCytomation, code no. K0699). The immunohistochemical protocol described above was optimized using ‘‘test’’ human tissue microarrays that contained selected lymphoid, epithelial and stromal tissues (i.e. spleen, colonic mucosa and kidney, endometrium, liver and uterine smooth muscle (code: CHTN2002 1). These arrays were used to titrate immunohistochemical assay parameters and antibody dilutions prior to use of the more comprehensive tissue microarrays. Three comprehensive tissue microarray sections were labeled with each FXYD antibody and all the studies were carried out with the CHTN2002N1 array; consecutive sections cut from a single master block on which each tissue type was represented in quadruplicate. The reproducibility of immunolabeling for each tissue type within the same section and between different arrays was excellent. 2.4. Data acquisition and analysis The stained TMA slides were independently scored in a double-blind fashion by two of the authors (AM, PM-V). The method of scoring involved visual histomorphometric analysis. The intensity of FXYD1 and FXYD2 immunostaining was evaluated by means of a three point scale: 0, no expression; 1, low expression; 2, abundant expression. The cellular distribution (presence or absence of cell specific immunohistochemical staining) was assessed using a four point scale: , no expression; + , scattered cells; ++, groups of cells or +++, most cells expressed FXYD1 and FXYD2 proteins. The semi-quantitative basis for the scoring as low to abundant expression was based on the apparent intensity of the labeling of specific cells and not the percentage of the section surface which was positively labeled. Each observer independently reviewed and compared the arrays and recorded the data directly into a Microsoft Excel worksheet. After the two observers independently completed the analysis and semiquantitative scoring of the same slides, results were compared. Using this approach inter-observer variation in histomorphometric scoring was minimal and the majority of scores were identical. In cases where variation was found, the difference was never more than one unit. The semiquantitative method adopted here has been validated and widely adopted for numerous immunohistochemical studies in our laboratory (Mobasheri and Marples, 2004; Mobasheri et al., 2005a, 2005b, 2007). The data obtained was linked to a database of digital images captured using a Nikon Microphot-FX microscope fitted with a Nikon DXM1200 digital camera.
3. Results The results of the immunohistochemical and histomorphometric data obtained in this paper have been summarized in Table 1. 3.1. Expression of FXYD1 and FXYD2 FXYD1 was highly expressed in the heart but found in moderate quantities in other cardiovascular tissues. Gastrointestinal, hepatic and pancreatobiliary systems expressed high levels of FXYD1. In the male genital system FXYD1 expression was detected in seminiferous tubules and seminal vesicles. In the
11
female genital system FXYD1 was present in the secretory endometrium of the uterus. High expression was also observed in the choroid plexus, amniotic membranes, placenta, salivary glands of the parotid, the epithelium of the breast, and the adrenal gland. Skeletal muscle and smooth muscles of the intestine and uterus also stained positively for FXYD1. Expression in the kidney, bladder, placenta and amniotic membranes was high but FXYD1 was not highly expressed in the central and peripheral nervous systems except in the cerebellar cortex (purkinje/granular layer) and ependymal cells. The central nervous system has also been reported to express other FXYD isoforms including FXYD7 (Beguin et al., 2002; Crambert et al., 2003), FXYD6 ((Adams et al., 1995; Sweadner and Rael, 2000); see Table 1) and a novel phospholemman-like protein named phosphohippolin (Yamaguchi et al., 2001). FXYD1 was also absent from the respiratory, lymphoid, soft and calcified tissues (but present in some cells in articular cartilage), adipose tissue and most reproductive tissues studied. The FXYD1 expression data has been summarized in Fig. 2 and Table 1. Expression of FXYD2, the g subunit of Na, K-ATPase was most abundant in the kidney (see below) but it was also detected in several other tissues from which FXYD2 has been cloned (i.e. colon, stomach, pancreas, liver and heart). FXYD2 was also detected in the antral and oxyntic gastric mucosa, in the epididymis, salivary glands, secretory epithelium of the breast and the endometrium. Expression of FXYD2 was relatively high in amniotic membranes, placenta, choroid plexus and adrenal medulla but lower in uterine smooth muscle and in the transitional epithelium of the urinary bladder. FXYD2 was also observed in the liver and colon (data not shown). The strongest FXYD2 immunostaining was observed in the distal renal tubule of the kidney (Figs. 3 and 4), the adrenal medulla, pancreas, salivary glands and amniotic membranes (Fig. 3). 3.2. Na, K-ATPase g isoform expression in human kidney The expression of the g subunit of Na, K-ATPase has been studied in the kidneys of rodents, pigs (Therien et al., 1997; Arystarkhova et al., 1999) and canines (Therien et al., 1997; Mobasheri et al., 2003) but no attempts have been made to immunolocalize the g subunit of Na, K-ATPase in normal human kidney. We conducted immunohistochemical studies on test TMAs and more comprehensive TMAs which samples of both normal human cortex and medulla were represented. The results shown in Fig. 4 confirm the limited expression of FXYD2 compared with the ‘‘a‘‘ subunits of Na, K-ATPase in human kidney. In the kidney the highest FXYD2 expression was located in the cortical distal collecting tubules and the cortical collecting ducts with lower levels in the cortical and medullary collecting ducts.
4. Discussion In this study we used high density human TMAs for a semiquantitative immunohistochemical and histomorphometric analysis of FXYD protein expression in normal human tissues. The major findings of this study are as follows: (1) expression of FXYD1 and FXYD2 proteins was restricted in human tissues; (2) In some tissues FXYD1 and FXYD2 were uniformly expressed in all cells whereas in other tissues the cellular distribution was not uniform and restricted to groups of cells or scattered cells. For example in cardiac muscle, amniotic membranes and placental villi FXYD1 was uniformly distributed in all cells across the tissue (except for connective tissue). In contrast the cellular distribution of FXYD1 and FXYD2 in the pancreas and salivary glands was
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Fig. 2. Immunohistochemical staining of FXYD1, phospholemman in selected human tissues. Phospholemman expression was detected in the heart (A), stomach (B, C), gallbladder (D), liver (E), pancreas (F), salivary glands (G), epithelium of the mammary gland (H), skeletal muscle (I), uterine smooth muscle (J), amniotic membranes (K), placenta (L), choroid plexus (M), kidney cortex and medulla (N, O), and transitional epithelium of the urinary bladder (P). The most distinctive phospholemman immunostaining was observed in the heart, stomach, liver, placental villi and kidney. Original magnification for each panel: 200 (approx.). Solid bars represent 100 mm.
restricted to groups of cells and in some tissues only scattered cells were positively stained (i.e. gastric mucosa); (3) the expression of the protein products of the FXYD1 and FXYD2 genes was not limited to the kidney; (4) FXYD2 was expressed in the adrenal gland; (5) other than the kidney the only tissues that exhibited abundant expression of both FXYD1 and FXYD2 were the choroid plexus, placental villi, amniotic membranes, cardiac muscle, gastric mucosa.
4.1. Distribution of phospholemman, FXYD1 The original study that proposed the name ‘‘phospholemman’’ for FXYD1 highlighted the fact that it is a substrate for cAMPdependent protein kinase A and protein kinase C, identified the protein’s location within the plasma membrane and its characteristic multiple phosphorylation sites and revealed its abundance in the myocardium (Palmer et al., 1991). This study confirms that FXYD1 is highly expressed in human cardiac and skeletal muscles which suggests this FXYD isoform plays a role in striated muscle contraction (Chen et al., 1997). The high FXYD1 expression levels observed with a1/b1 isoenzymes of Na, K-ATPase in the heart and skeletal muscle (Crambert et al., 2002) correlates well with the high Na, K-ATPase expression in these tissues and suggests that FXYD1 plays a functional role in
cardiac and skeletal muscle contraction. This has been suggested by other investigators in mammals and dogfish (Chen et al., 1997; Crambert and Geering, 2003; Schuurmans Stekhoven et al., 2003). Further evidence for the involvement of FXYD1 in cardiac muscle function comes from recent studies in rat ventricular myocytes where FXYD1 over-expression modulates myocyte contractility by inhibiting the Na + /Ca2 + exchanger (Zhang et al., 2003) and altering [Ca2 + ]i transients (Song et al., 2002). There was, however, little or no expression of FXYD1 in the blood vessels examined or the two other smooth muscles (intestinal and uterine) studied. Although we were able to detect FXYD1 protein expression in the myocardium, immunostaining in aortic tissue was absent. This does not imply that blood vessels lack these proteins; there may simply be much lower expression in blood vessels compared with myocardial tissues. In the urinary system (i.e. kidneys and urinary bladder) the expression of FXYD1 and the g and a subunits of Na, K-ATPase were also high: FXYD1 is a major target of protein kinases and it has recently been reported to be located in the juxtaglomerular apparatus where it might be involved in tubuloglomerular feedback (Wetzel and Sweadner, 2003). In this study we found that FXYD1 expression was also high in the human gastrointestinal system (antral and oxyntic gastric mucosa, small intestine, and colon). In particular FXYD1 expression in the rectum and colon confirms recent data from the
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Fig. 3. Immunohistochemical staining of FXYD2, the g subunit of Na, K-ATPase in selected human tissues. FXYD2 was detected in the heart (A), antral and oxyntic gastric mucosa (B, C), epididymis (D), pancreas (E), salivary glands (F), breast epithelium (G), secretory endometrial epithelium (H), uterine smooth muscle (I), adrenal medulla (J), amniotic membranes (K), placenta (L), choroid plexus (M), kidney cortex and medulla (N, O), and transitional epithelium of the urinary bladder (P). FXYD2 expression was also observed in the liver and colon (data not shown). The most striking FXYD2 immunostaining was observed in the pancreas, salivary glands, adrenal medulla, amniotic membranes and the distal nephron in the kidney. Original magnification for each panel: 200 (approx.). Solid bars represent 100 mm.
rectal glands of the shark Squalus acanthias (Mahmmoud et al., 2000). We speculate that FXYD1 associated with Na, K-ATPase may play an important role in regulating nutrient uptake in the gastrointestinal system. Different segments of the gut are involved in solute and fluid absorption and secretion. This may necessitate a diverse set of transporters with different kinetic properties adding to the complexity of sodium and osmolyte transport regulation in the gut. For example, in the small intestine sodium dependent co-transporters such as SGLT-1 are involved in the apical uptake of glucose from the lumen of the gut. FXYD1 in the basolateral membrane of enterocytes may be an important regulator of Na, K-ATPase function and sodium dependent nutrient transport systems. This could be one of the reasons for the complexity of sodium, nutrient and osmolyte transport in the gut. The apical presence of FXYD1 in the human choroid plexus (the organ that secretes CSF in the ventricles) confirms recent studies in the rat central nervous system where FXYD1 was found to be particularly enriched in choroid plexus, where it co-localized with Na, K-ATPase in the apical membrane (Kajita and Brown, 1997; Feschenko et al., 2003). Another potential function of FXYD1 as a separate channel entity is modulation of taurine transport (Morales-Mulia et al., 2000). Taurine is an important organic osmolyte which has been
shown to be critical for brain adaptation to hypo-osmolarity and neuronal and glial cell volume regulation (Pasantes-Morales et al., 2000). The presence of FXYD1 in the cerebellum and choroid plexus suggests that it may modulate taurine flux in the brain (Moran et al., 2001). Collectively the results presented here confirm recent reports of FXYD1 expression in rat tissues and suggest that FXYD1 may not only be involved in the physiological regulation of contractile tissues and urinary epithelial tissues but also in regulating Na, K-ATPase, other ATPases or ion transporters in reproductive (i.e. epididymis; (Xu et al., 2003)), gastrointestinal, hepatic and pancreatobiliary systems where additional complexity of sodium and osmolyte transport regulation will be found.
4.2. Distribution of the g subunit of Na, K-ATPase, FXYD2 The expression of FXYD2 has been studied in the kidneys of several species including rodents, pigs and canines (Therien et al., 1997; Arystarkhova et al., 1999; Mobasheri et al., 2003) but this is the only study that has successfully immunolocalized FXYD2 in the normal human kidney. The expression of FXYD2 was particularly high in cortical distal nephron segments where the epithelial sodium channel (ENaC) is also thought to be located
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Fig. 4. Immunohistochemical staining of FXYD2, the g subunit of Na, K-ATPase and the ‘‘a‘‘ subunits of Na, K-ATPase in human kidney. In these experiments alkaline phosphatase conjugated secondary antibody was used with the Fast Red Substrate System. Panels A and C represent a subunit expression in human renal cortex and panels B and D represent a subunit expression in the renal medulla. Panels E and G represent FXYD2 expression in human renal cortex and panels F and H represent FXYD2 expression in the renal medulla. The expression of the g subunit was less abundant than the a subunit and the distribution of FXYD2 appeared to be limited to the distal nephron (i.e. collecting tubule and the distal convoluted tubule). Original magnification for each panel: 200 (approx.).
(Loffing and Kaissling, 2003). It is plausible that the restricted presence of FXYD2 in the distal nephron may be important for modulating basolateral Na, K-ATPase and the trans-epithelial sodium re-absorption mediated by the concerted action of ENaC and Na, K-ATPase in principal cells. Naturally, it is essential to adapt TMAs for double immunofluorescent labeling (using specific markers of different nephron segments or cell types such as principal cells and intercalated cells) before such a conclusion
can be reached. Therefore the presence of FXYD2 in human kidney is consistent with a major physiological role for the g isoform of Na, K-ATPase in modulating Na, K-ATPase in the renal epithelium (Pu et al., 2001; Farman et al., 2003; PihakaskiMaunsbach et al., 2003). Previous immunocytochemical studies in rat kidney have shown that the FXYD2 and CHIF (FXYD4) are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of
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the Na, K-ATPase activity in the collecting duct (PihakaskiMaunsbach et al., 2003). Our results also confirmed that FXYD2 is expressed in several other tissues from which the cDNA encoding the g protein has been cloned1; i.e. colon, stomach, pancreas, liver and heart (Kim et al., 1997). Expression of FXYD2 in the heart is non-classical and intriguing. Previous studies have used a polyclonal anti-g antiserum to define the tissue specificity of FXYD2 and to determine whether it has a functional role and used western blot analysis to show that the protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells or transfected HeLa cells (Therien et al., 1997). However, the immunohistochemical studies presented here and a recent review of the molecular and functional studies of FXYD2 suggest that it may be expressed in human cardiac muscle (Therien et al., 2001). There may well be differences between the FXYD isoform profiles of human, rodent, canine and porcine cardiac muscle since it is already known that not all Na, K-ATPase isoforms are expressed in human, rodent and guinea pig heart (McDonough et al., 1996). The heart is also a major target of the renin-angiotensinaldosterone system which not only regulates collagen turnover and fibrous tissue formation, but also sodium transport (Delcayre and Swynghedauw, 2002). Until relatively recently it was widely believed that the mineralocorticoid hormone aldosterone (produced in the adrenal cortex) acted exclusively on ion transporting epithelia to promote sodium retention and potassium excretion. However, it is now known that aldosterone also acts on nonepithelial tissues, such as heart and blood vessels (Rocha and Funder, 2002). Therefore expression of FXYD2 in the human heart is perhaps not surprising given the abundance of mineralocorticoid receptors and potential for endocrine regulation of sodium transport. Further experiments are clearly necessary to determine the physiological significance of FXYD2 expression in the human myocardium where three alpha isoforms of Na, K-ATPase are known to be present. FXYD2 protein was also detected in the antral and oxyntic gastric mucosa, in the epididymis, salivary glands, secretory epithelium of the breast and the uterine endometrium. It was also highly abundant in the transport active epithelium of the amnion, placenta and choroid plexus. FXYD2 and FXYD1 were both expressed in the amniotic membrane and placental villi where they may modulate Na, K-ATPase function. In conclusion, the results of this study confirm our hypothesis that the distribution of FXYD1 and FXYD2 proteins in human tissues is more widespread than is apparent from the current literature. These FXYD proteins may associate with Na, K-ATPase in various epithelial and cardiovascular tissues and influence its catalytic properties. Tissue microarray technology will be a useful tool for determining the tissue distribution of novel members of the FXYD gene family as antibodies to these proteins gradually become available.
Acknowledgements This study was funded by the Wellcome Trust UK (Grant number 065559/Z/01/). We express our gratitude to Dr. S.J.D. Karlish (Weizmann Institute of Science, Rehovot, Israel) for providing the polyclonal antibodies to phospholemman and the g subunit of Na, K-ATPase. We are also grateful to Dr. M. Takahashi and Dr. M.W. McEnery for the pan a specific 1 http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html, NCBI AceView, April 2009.
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monoclonal mAb 9A7. We would also like to acknowledge Dr. Cecilia M. Canessa (Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut) and Dr. Nicolette Farman (INSERM Research Unit 478, Faculte´ Xavier Bichat, Paris) for critical comments on the manuscript. References Adams, M.D., Kerlavage, A.R., Fleischmann, R.D., Fuldner, R.A., Bult, C.J., Lee, N.H., Kirkness, E.F., Weinstock, K.G., Gocayne, J.D., White, O., et al., 1995. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377, 3–174. Arystarkhova, E., Wetzel, R.K., Asinovski, N.K., Sweadner, K.J., 1999. The gamma subunit modulates Na(+ ) and K(+ ) affinity of the renal Na,K-ATPase. J. Biol. Chem. 274, 33183–33185. Attali, B., Latter, H., Rachamim, N., Garty, H., 1995. A corticosteroid-induced gene expressing an ‘‘IsK-like’’ K+ channel activity in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 92, 6092–6096. 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Contents lists available at ScienceDirect
Annals of Anatomy journal homepage: www.elsevier.de/aanat
Differential cellular expression of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in normal human tissues: A study using high density human tissue microarrays Rachel V. Floyd a, Susan Wray a, Pablo Martı´n-Vasallo b, Ali Mobasheri c,n a
Physiological Laboratory, Department of Physiology, School of Biomedical Sciences, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 3BX, UK Laboratorio de Biologı´a del Desarrollo, Departamento de Bioquı´mica y Biologı´a Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain c Musculoskeletal Research Group, Division of Veterinary Medicine, School of Veterinary Medicine and Science, Institute of Clinical Research and the Faculty of Medicine and Health Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK b
a r t i c l e in fo
abstract
Article history: Received 13 April 2009 Received in revised form 3 August 2009 Accepted 15 September 2009
FXYD proteins have been proposed to function as regulators of Na, K-ATPase function by lowering affinities of the system for potassium and sodium. However, their distribution in normal human tissues has not been studied. We have therefore used immunohistochemistry and semi-quantitative histomorphometric analysis to determine the relative expression at the protein level and distribution of FXYD1 (phospholemman) and FXYD2 (g subunit of Na, K-ATPase) in human Tissue MicroArrays (TMAs). Expression of FXYD1 was abundant in heart, kidney, placenta, skeletal muscle, gastric and anal mucosa, small intestine and colon. Lower FXYD1 expression was detected in uterine, intestinal and bladder smooth muscle, choroid plexus, liver, gallbladder, spleen, breast, prostate and epididymis. The tissue distribution of FXYD2 was less extensive compared to that of FXYD1. There was an abundant expression in kidney and choroid plexus and moderate expression in placenta, amniotic membranes, breast epithelium, salivary glands, pancreas and uterine endometrium. Weaker FXYD2 expression was detected in the adrenal medulla, liver, gallbladder, bladder and pancreas. The common denominator in the distribution of FXYD1 and FXYD2 was expression in highly active transport epithelia of the kidney, choroid plexus, placenta and salivary glands. This study reveals, in human tissues, the specific expression of FXYD proteins, which may associate with Na, K-ATPase in selected cell types and modulate its catalytic properties. & 2009 Elsevier GmbH. All rights reserved.
Keywords: FXYD1 (phospholemman) FXYD2 Tissue microarray Immunohisto chemistry Histomorphometric analysis
1. Introduction It is becoming increasingly recognized that small single-span membrane proteins (30 + amino acids) play important physiological roles by regulating the activity of larger macromolecular complexes that form ion permeant channels, transporters and ATPase pumps. For example, phospholamban regulates the properties of SERCA2a, the Ca-ATPase of cardiac sarcoplasmic reticulum, and its homolog sarcolipin regulates the SERCA1a CaATPase isoform of fast-twitch skeletal muscle (Odermatt et al., 1998; Simmerman and Jones, 1998). The FXYD gene family of ion transport regulators contains seven small single-span membrane proteins that share a 35-amino acid signature sequence domain, beginning with the sequence PFXYD and containing 7 invariant and 6 highly conserved amino acids (Sweadner and Rael, 2000) (see Table 1). The FXYD proteins have been proposed to function
n
Corresponding author. Tel.: + 44 115 951 6449; fax: + 44 115 951 6440. E-mail address: [email protected] (A. Mobasheri).
0940-9602/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.aanat.2009.09.003
as novel tissue- and isoform-specific regulators of the plasma membrane Na, K-ATPase (Geering et al., 2003). Four of the FXYD proteins (FXYD1, FXYD2, FXYD4, FXYD7) have been shown to alter the activity of the Na, K-ATPase (Cornelius et al., 2001; Sweadner et al., 2003). Thus, the tissue- and isozyme-specific interaction of Na, K-ATPase with FXYD proteins may contribute to the proper handling of Na + and K + by Na, K-ATPase in physiological processes such as renal Na + -re-absorption, muscle contraction and neuronal excitability (Crambert and Geering, 2003). The protein products of the FXYD family have been predicted to exhibit ion channel or ion transport activity and localize in the plasma membrane. Messenger RNA transcripts encoding proteins of the FXYD family are abundant and widespread in fetal and adult mammalian tissues, particularly in epithelial tissues that perform bulk fluid and electrolyte transport including kidney, pancreas, colon, mammary glands, prostate, liver, lung and placenta. FXYD members are also abundantly expressed in cardiovascular and electrically excitable tissues (central and peripheral nervous system and various types of muscle). The best studied
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Table 1 Summary of the expression levels and cellular distribution of FXYD1 and FXYD2 in tissues represented on the CHTN2002N1 tissue microarray (http://faculty.virginia.edu/ chtn-tma/2002N1/CHTN2002normal%20TMA.html). System
Tissue
FXYD1
FXYD2
Cellular distribution
Staining intensity
Cellular distribution
Staining intensity
Cardiovascular system
aorta, smooth muscle heart, myocardium lymphatic endothelium small muscular artery (lung) small vein (intestine)
+++ ++
0 2 0 0 1
+++
0 1 0 0 0
Gastrointestinal system
esophagus, squamous mucosa gastric mucosa, antral gastric mucosa, oxyntic small intestine, mucosa colon, mucosa anus, mucosa
mucosa++ ++ + + +
1 1 2 1 1 0
++ ++ ++ ++
0 1 2 1 1 0
Genital system (male)
seminiferous tubules epididymis seminal vessicle prostate
+ +
0 1 1 0
+ ++
0 1 1 0
Hepatic (pancreatobiliary)
gallbladder liver pancreas
+++ +++ +
2 1 1
+++ ++
1 0 1
Oral, salivary & nasal
salivary gland (parotid) tonsil, squamous epithelium
++
1 0
+
2 0
Skin
skin, squamous epithelium subepidermal tissue
0 0
0 0
Breast
breast, epithelium
++
1
++
2
Genital system (female)
ectocervix endocervix endometrium, secretory fallopian tube
+++
0 0 1 0
+ +++
0 1 2 0
Endocrine system
adrenal gland, cortex adrenal gland, medulla parathyroid adenoma pituitary, anterior pituitary, posterior thyroid
+++
1 0 0 0 0 0
+ +
1 1 0 0 0 0
Soft tissue
adipose tissue, breast
0
0
Cartilage
cartilage, articular
+
1
0
Soft tissue
cartilage, bronchial
0
0
Soft tissue (muscular)
skeletal muscle smooth muscle, intestine smooth muscle, uterus
+++ ++
1 0 1
++
0 0 1
Synovium
synovium
0
0
Genital (female)
ovary, ovary, ovary, ovary,
+++ +++ +++ +++
1 1 1 1
++ +++ ++ ++
1 2 1 1
Placenta
amniotic membrane placenta, villi
+++ +++
1 2
+++ ++
2 1
Peripheral nervous system
autonomic ganglia & nerves, intestinal peripheral nerve
0
0
0
0
Central nervous system
cerebral cortex cerebellar cortex, purkinje/ granular layer choroid plexus ependymal cells meninges motor neurons (spinal cord) white matter (subcortical) hippocampus
+ +
1 1
0 0
+++ + + +
2 1 0 1 0 1
+
2 0 0 0 0 0
0 0
0 0
Lymphoid system
primary oocytes corpus luteum epithelium stroma
lymph node mucosa associated lymphoid tissue, appendix
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Table 1 (continued ) System
Tissue
FXYD1
FXYD2
Cellular distribution
Staining intensity
Cellular distribution
Staining intensity
spleen thymus tonsil
0 0 0
0 0 0
Respiratory system
alveoli bronchus, epithelium
0 0
0 0
Urological system
kidney, cortex kidney, medulla bladder, transitional epithelium
++ ++ +++
2 2 2
++ ++ +++
2 2 2
Key
Key
+ ++
No expression Scattered cells Groups of cells
+++
Most cells
0 1 2
No expression Low expression Abundant expression
The cellular distribution was assessed using a four point scale: , no expression; + , scattered cells; ++, groups of cells; +++, most cells expressed FXYD1 and FXYD2 proteins. The intensity of FXYD1 and FXYD2 immunostaining was evaluated by means of a three point scale: 0, no expression; 1, low expression; 2, abundant expression.
members of this gene family are FXYD1 and FXYD2. FXYD1 also known as phospholemman is a plasma membrane substrate for several kinases, including protein kinase A, protein kinase C, NIMA (Never in mitosis, gene A) kinase, and myotonic dystrophy kinase (Palmer et al., 1991; Moorman et al., 1992; Lu et al., 1994). It is thought to form an ion channel or regulate ion channel activity. FXYD2 is the g subunit of Na, K-ATPase (also known as HOMG2, ATP1G1 or MGC12372) and assembles with the a and b subunits of the Na, K-ATPase and forms an association tight enough to survive detergent extraction and extensive proteolysis (Or et al., 1996; Therien et al., 2001). FXYD7 is known to be expressed in the central nervous system and has been suggested to be a brainspecific regulator of Na, K-ATPase (Crambert and Geering, 2003; Crambert et al., 2003; Geering et al., 2003). Other FXYD family members are less well studied particularly FXYD3 or MAT8 (mammary tumor marker) (Morrison et al., 1995) FXYD4 or CHIF (channel-inducing factor) (Attali et al., 1995), FXYD5 or RIC (related to ion channel) (Fu and Kamps (1997)) and the relatively uncharacterized FXYD6. Although FXYD1-5 have all been shown to induce ion channel activity in experimental expression systems (Attali et al., 1995; Morrison et al., 1995; Kowdley et al., 1997; Minor et al., 1998) their distribution in normal rodent or human tissues has not been studied using high throughput immunohistochemistry. The recent proliferation of Tissue MicroArray (TMA) technology allows screening of large numbers of normal and tumor tissue specimens for gene and protein expression information and for discovering novel diagnostic and prognostic correlations. We have taken advantage of TMA technology to test the hypothesis that the distribution of FXYD1 and FXYD2 proteins in the human body is more widespread than the cardiovascular and renal systems. Nerve and muscle cells require low Na + affinity Na, K-ATPase pumps capable of rapid and efficient extrusion of intracellular Na + during periods of intense ion transport activity. The same principle may be applied to transport epithelia responsible for bulk Na + and K + transport during fundamental activities such as secretion and absorption. Therefore, in these cells/tissues co-expression of small ion transport regulators (i.e. FXYD proteins) along with the a and b subunits of Na, K-ATPase may provide the transport system with a kinetic advantage by allowing the pump to increase its rate in response to sudden and rapid rises in intracellular Na + . To test this idea we used immunohistochemistry and semi-quantitative histomorphometric
analysis to study the distribution and relative abundance of the FXYD1 and FXYD2 proteins in high density human TMAs.
2. Materials and methods 2.1. Chemicals All chemicals used were molecular biology grade and purchased from Sigma/Aldrich and Sigma Biosciences (Poole, Dorset, UK). Materials for immunohistochemistry were purchased from Vector Laboratories (Peterborough, UK) and DakoCytomation (Ely, Cambridgeshire, UK). Antibodies Polyclonal PLM-antibody, produced against a peptide CRSSIRRLSTRRR of the C terminus of rat PLM recognizes FXYD1 (Crambert et al., 2002). Polyclonal gC33 antiserum was raised against the 10-residue C terminus of the g variant (Kuster et al., 2000). The antibodies were diluted 1:300 before use. Both antisera were kind gifts of Dr. S.J.D. Karlish (Weizmann Institute of Science, Rehovoth, Israel). 2.2. High density human Tissue MicroArrays (TMAs) Tissue MicroArrays (TMAs) of formalin fixed paraffin embedded human tissues (Fig. 1) were obtained from the Cooperative Human Tissue Network (CHTN) of The National Cancer Institute (NCI), the National Institutes of Health, Bethesda, MD, USA (http://faculty.virginia.edu/chtn-tma/home.html). Medical ethics committee approval was not required for these studies. The TMAs contained 66 anonymized samples of non-neoplastic adult tissues obtained from surgical resection specimens, fixed within one hour of surgical removal from the donors. The central nervous system tissues on the TMAs were obtained from autopsy specimens within 36 hours of death. The tissues represented on the TMAs included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary and nasal, mammary, endocrine, genital tract, central and peripheral nervous system, urinary tract, skin, cartilage and synovium. In view of the extreme
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Fig. 1. Representative Tissue MicroArray (TMA) slide consisting of 0.6 mm diameter spots of 66 normal tissue types present in the human body. The slides used throughout this study were codenamed CHTN2002N1 and obtained from the Cooperative Human Tissue Network of The National Cancer Institute, the National Institutes of Health. Higher magnification views in panel B illustrate (clockwise) hematoxylin and eosin stained spots of kidney medulla, the mucosa of the small intestine, skeletal muscle and parotid salivary gland. Solid bars represent 100 mm. The tissues cores represented on the microarray are classified by the system from which they originate (C) and are grouped together in distinct square blocks. Each cluster of spots represents tissues from a particular system (i.e. cardiovascular, urinary, etc.). For a complete list of tissues refer to http://faculty.virginia.edu/chtn-tma/2002N1/CHTN2002N1.htm.
cellular heterogeneity of complex tissues such as the brain and the wide diversity of endocrine glands, it must be pointed out that these TMAs are not a complete representation of the human body. Further details and layout of the CHTN normal human tissue TMA may be found on the CHTN website: http://faculty.virginia.edu/ chtn-tma/2002N1/CHTN2002N1X.xls.
2.3. Immunohistochemical protocol The TMAs were heated at 60 1C for 15 min to improve tissue adhesion to the charged glass slides (Fisher Plus). Prior to immunostaining, TMA slides were deparaffinized in xylene for 20 min to remove embedding media and washed in absolute ethanol for 3 min. The TMAs were gradually rehydrated in a series of alcohol baths (96%, 85% and 50%) and placed in distilled water for 5 min. Endogenous peroxidase was blocked for 1 hour in a 97% methanol solution containing 3% hydrogen peroxide and 0.01% sodium azide. Following thrice washing in phosphate buffered saline (PBS) the TMAs were subjected to antigen retrieval by incubating with 0.5% sodium dodecyl sulfate (SDS) in PBS for
10 min (Brown et al., 1996). Excess SDS was removed by incubating the TMAs for 5 min in PBS. To prevent non-specific antibody binding the TMAs were incubated for one hour at room temperature (RT) with 20% normal goat serum (NGS) in PBS containing 1% bovine serum albumin and 0.01% sodium azide. Slides were incubated overnight at 4 1C with antisera. After 24 hrs at 4 1C the slides were washed 3 times for 5 min in PBS before incubating with horseradish peroxidase labeled polymer conjugated to affinity purified goat anti-rabbit and goat anti-mouse immunoglobulins (DAKO code no. K4010) for 30 minutes (RT). The sections were washed 3 times for 5 min in PBS before applying liquid DAB+ Chromogen (DAKO; 3,30 -diaminobenzidine chromogen solution) for up to 30 s. The development of the brown colored reaction was stopped by rinsing in distilled water. The stained slides were immersed for 5 min in a bath of aqueous hematoxylin (DakoCytomation, code no. S3309) to counterstain cell nuclei. Finally the slides were washed for 5 min in running water and dehydrated in a series of graded ethanol baths before rinsing in 3 xylene baths and mounting in DPX (BDH laboratories). In some experiments (see immunohistochemical data shown in Fig. 4) the slides were incubated with alkaline phosphatase
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labeled polymer conjugated to affinity purified goat anti-rabbit and goat anti-mouse immunoglobulins instead of horseradish peroxidase labeled polymer. In these experiments the Fast Red Substrate System (tablets containing naphthol phosphate, fast red and levamisole) was used as a chromogen (DakoCytomation, code no. K0699). The immunohistochemical protocol described above was optimized using ‘‘test’’ human tissue microarrays that contained selected lymphoid, epithelial and stromal tissues (i.e. spleen, colonic mucosa and kidney, endometrium, liver and uterine smooth muscle (code: CHTN2002 1). These arrays were used to titrate immunohistochemical assay parameters and antibody dilutions prior to use of the more comprehensive tissue microarrays. Three comprehensive tissue microarray sections were labeled with each FXYD antibody and all the studies were carried out with the CHTN2002N1 array; consecutive sections cut from a single master block on which each tissue type was represented in quadruplicate. The reproducibility of immunolabeling for each tissue type within the same section and between different arrays was excellent. 2.4. Data acquisition and analysis The stained TMA slides were independently scored in a double-blind fashion by two of the authors (AM, PM-V). The method of scoring involved visual histomorphometric analysis. The intensity of FXYD1 and FXYD2 immunostaining was evaluated by means of a three point scale: 0, no expression; 1, low expression; 2, abundant expression. The cellular distribution (presence or absence of cell specific immunohistochemical staining) was assessed using a four point scale: , no expression; + , scattered cells; ++, groups of cells or +++, most cells expressed FXYD1 and FXYD2 proteins. The semi-quantitative basis for the scoring as low to abundant expression was based on the apparent intensity of the labeling of specific cells and not the percentage of the section surface which was positively labeled. Each observer independently reviewed and compared the arrays and recorded the data directly into a Microsoft Excel worksheet. After the two observers independently completed the analysis and semiquantitative scoring of the same slides, results were compared. Using this approach inter-observer variation in histomorphometric scoring was minimal and the majority of scores were identical. In cases where variation was found, the difference was never more than one unit. The semiquantitative method adopted here has been validated and widely adopted for numerous immunohistochemical studies in our laboratory (Mobasheri and Marples, 2004; Mobasheri et al., 2005a, 2005b, 2007). The data obtained was linked to a database of digital images captured using a Nikon Microphot-FX microscope fitted with a Nikon DXM1200 digital camera.
3. Results The results of the immunohistochemical and histomorphometric data obtained in this paper have been summarized in Table 1. 3.1. Expression of FXYD1 and FXYD2 FXYD1 was highly expressed in the heart but found in moderate quantities in other cardiovascular tissues. Gastrointestinal, hepatic and pancreatobiliary systems expressed high levels of FXYD1. In the male genital system FXYD1 expression was detected in seminiferous tubules and seminal vesicles. In the
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female genital system FXYD1 was present in the secretory endometrium of the uterus. High expression was also observed in the choroid plexus, amniotic membranes, placenta, salivary glands of the parotid, the epithelium of the breast, and the adrenal gland. Skeletal muscle and smooth muscles of the intestine and uterus also stained positively for FXYD1. Expression in the kidney, bladder, placenta and amniotic membranes was high but FXYD1 was not highly expressed in the central and peripheral nervous systems except in the cerebellar cortex (purkinje/granular layer) and ependymal cells. The central nervous system has also been reported to express other FXYD isoforms including FXYD7 (Beguin et al., 2002; Crambert et al., 2003), FXYD6 ((Adams et al., 1995; Sweadner and Rael, 2000); see Table 1) and a novel phospholemman-like protein named phosphohippolin (Yamaguchi et al., 2001). FXYD1 was also absent from the respiratory, lymphoid, soft and calcified tissues (but present in some cells in articular cartilage), adipose tissue and most reproductive tissues studied. The FXYD1 expression data has been summarized in Fig. 2 and Table 1. Expression of FXYD2, the g subunit of Na, K-ATPase was most abundant in the kidney (see below) but it was also detected in several other tissues from which FXYD2 has been cloned (i.e. colon, stomach, pancreas, liver and heart). FXYD2 was also detected in the antral and oxyntic gastric mucosa, in the epididymis, salivary glands, secretory epithelium of the breast and the endometrium. Expression of FXYD2 was relatively high in amniotic membranes, placenta, choroid plexus and adrenal medulla but lower in uterine smooth muscle and in the transitional epithelium of the urinary bladder. FXYD2 was also observed in the liver and colon (data not shown). The strongest FXYD2 immunostaining was observed in the distal renal tubule of the kidney (Figs. 3 and 4), the adrenal medulla, pancreas, salivary glands and amniotic membranes (Fig. 3). 3.2. Na, K-ATPase g isoform expression in human kidney The expression of the g subunit of Na, K-ATPase has been studied in the kidneys of rodents, pigs (Therien et al., 1997; Arystarkhova et al., 1999) and canines (Therien et al., 1997; Mobasheri et al., 2003) but no attempts have been made to immunolocalize the g subunit of Na, K-ATPase in normal human kidney. We conducted immunohistochemical studies on test TMAs and more comprehensive TMAs which samples of both normal human cortex and medulla were represented. The results shown in Fig. 4 confirm the limited expression of FXYD2 compared with the ‘‘a‘‘ subunits of Na, K-ATPase in human kidney. In the kidney the highest FXYD2 expression was located in the cortical distal collecting tubules and the cortical collecting ducts with lower levels in the cortical and medullary collecting ducts.
4. Discussion In this study we used high density human TMAs for a semiquantitative immunohistochemical and histomorphometric analysis of FXYD protein expression in normal human tissues. The major findings of this study are as follows: (1) expression of FXYD1 and FXYD2 proteins was restricted in human tissues; (2) In some tissues FXYD1 and FXYD2 were uniformly expressed in all cells whereas in other tissues the cellular distribution was not uniform and restricted to groups of cells or scattered cells. For example in cardiac muscle, amniotic membranes and placental villi FXYD1 was uniformly distributed in all cells across the tissue (except for connective tissue). In contrast the cellular distribution of FXYD1 and FXYD2 in the pancreas and salivary glands was
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Fig. 2. Immunohistochemical staining of FXYD1, phospholemman in selected human tissues. Phospholemman expression was detected in the heart (A), stomach (B, C), gallbladder (D), liver (E), pancreas (F), salivary glands (G), epithelium of the mammary gland (H), skeletal muscle (I), uterine smooth muscle (J), amniotic membranes (K), placenta (L), choroid plexus (M), kidney cortex and medulla (N, O), and transitional epithelium of the urinary bladder (P). The most distinctive phospholemman immunostaining was observed in the heart, stomach, liver, placental villi and kidney. Original magnification for each panel: 200 (approx.). Solid bars represent 100 mm.
restricted to groups of cells and in some tissues only scattered cells were positively stained (i.e. gastric mucosa); (3) the expression of the protein products of the FXYD1 and FXYD2 genes was not limited to the kidney; (4) FXYD2 was expressed in the adrenal gland; (5) other than the kidney the only tissues that exhibited abundant expression of both FXYD1 and FXYD2 were the choroid plexus, placental villi, amniotic membranes, cardiac muscle, gastric mucosa.
4.1. Distribution of phospholemman, FXYD1 The original study that proposed the name ‘‘phospholemman’’ for FXYD1 highlighted the fact that it is a substrate for cAMPdependent protein kinase A and protein kinase C, identified the protein’s location within the plasma membrane and its characteristic multiple phosphorylation sites and revealed its abundance in the myocardium (Palmer et al., 1991). This study confirms that FXYD1 is highly expressed in human cardiac and skeletal muscles which suggests this FXYD isoform plays a role in striated muscle contraction (Chen et al., 1997). The high FXYD1 expression levels observed with a1/b1 isoenzymes of Na, K-ATPase in the heart and skeletal muscle (Crambert et al., 2002) correlates well with the high Na, K-ATPase expression in these tissues and suggests that FXYD1 plays a functional role in
cardiac and skeletal muscle contraction. This has been suggested by other investigators in mammals and dogfish (Chen et al., 1997; Crambert and Geering, 2003; Schuurmans Stekhoven et al., 2003). Further evidence for the involvement of FXYD1 in cardiac muscle function comes from recent studies in rat ventricular myocytes where FXYD1 over-expression modulates myocyte contractility by inhibiting the Na + /Ca2 + exchanger (Zhang et al., 2003) and altering [Ca2 + ]i transients (Song et al., 2002). There was, however, little or no expression of FXYD1 in the blood vessels examined or the two other smooth muscles (intestinal and uterine) studied. Although we were able to detect FXYD1 protein expression in the myocardium, immunostaining in aortic tissue was absent. This does not imply that blood vessels lack these proteins; there may simply be much lower expression in blood vessels compared with myocardial tissues. In the urinary system (i.e. kidneys and urinary bladder) the expression of FXYD1 and the g and a subunits of Na, K-ATPase were also high: FXYD1 is a major target of protein kinases and it has recently been reported to be located in the juxtaglomerular apparatus where it might be involved in tubuloglomerular feedback (Wetzel and Sweadner, 2003). In this study we found that FXYD1 expression was also high in the human gastrointestinal system (antral and oxyntic gastric mucosa, small intestine, and colon). In particular FXYD1 expression in the rectum and colon confirms recent data from the
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Fig. 3. Immunohistochemical staining of FXYD2, the g subunit of Na, K-ATPase in selected human tissues. FXYD2 was detected in the heart (A), antral and oxyntic gastric mucosa (B, C), epididymis (D), pancreas (E), salivary glands (F), breast epithelium (G), secretory endometrial epithelium (H), uterine smooth muscle (I), adrenal medulla (J), amniotic membranes (K), placenta (L), choroid plexus (M), kidney cortex and medulla (N, O), and transitional epithelium of the urinary bladder (P). FXYD2 expression was also observed in the liver and colon (data not shown). The most striking FXYD2 immunostaining was observed in the pancreas, salivary glands, adrenal medulla, amniotic membranes and the distal nephron in the kidney. Original magnification for each panel: 200 (approx.). Solid bars represent 100 mm.
rectal glands of the shark Squalus acanthias (Mahmmoud et al., 2000). We speculate that FXYD1 associated with Na, K-ATPase may play an important role in regulating nutrient uptake in the gastrointestinal system. Different segments of the gut are involved in solute and fluid absorption and secretion. This may necessitate a diverse set of transporters with different kinetic properties adding to the complexity of sodium and osmolyte transport regulation in the gut. For example, in the small intestine sodium dependent co-transporters such as SGLT-1 are involved in the apical uptake of glucose from the lumen of the gut. FXYD1 in the basolateral membrane of enterocytes may be an important regulator of Na, K-ATPase function and sodium dependent nutrient transport systems. This could be one of the reasons for the complexity of sodium, nutrient and osmolyte transport in the gut. The apical presence of FXYD1 in the human choroid plexus (the organ that secretes CSF in the ventricles) confirms recent studies in the rat central nervous system where FXYD1 was found to be particularly enriched in choroid plexus, where it co-localized with Na, K-ATPase in the apical membrane (Kajita and Brown, 1997; Feschenko et al., 2003). Another potential function of FXYD1 as a separate channel entity is modulation of taurine transport (Morales-Mulia et al., 2000). Taurine is an important organic osmolyte which has been
shown to be critical for brain adaptation to hypo-osmolarity and neuronal and glial cell volume regulation (Pasantes-Morales et al., 2000). The presence of FXYD1 in the cerebellum and choroid plexus suggests that it may modulate taurine flux in the brain (Moran et al., 2001). Collectively the results presented here confirm recent reports of FXYD1 expression in rat tissues and suggest that FXYD1 may not only be involved in the physiological regulation of contractile tissues and urinary epithelial tissues but also in regulating Na, K-ATPase, other ATPases or ion transporters in reproductive (i.e. epididymis; (Xu et al., 2003)), gastrointestinal, hepatic and pancreatobiliary systems where additional complexity of sodium and osmolyte transport regulation will be found.
4.2. Distribution of the g subunit of Na, K-ATPase, FXYD2 The expression of FXYD2 has been studied in the kidneys of several species including rodents, pigs and canines (Therien et al., 1997; Arystarkhova et al., 1999; Mobasheri et al., 2003) but this is the only study that has successfully immunolocalized FXYD2 in the normal human kidney. The expression of FXYD2 was particularly high in cortical distal nephron segments where the epithelial sodium channel (ENaC) is also thought to be located
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Fig. 4. Immunohistochemical staining of FXYD2, the g subunit of Na, K-ATPase and the ‘‘a‘‘ subunits of Na, K-ATPase in human kidney. In these experiments alkaline phosphatase conjugated secondary antibody was used with the Fast Red Substrate System. Panels A and C represent a subunit expression in human renal cortex and panels B and D represent a subunit expression in the renal medulla. Panels E and G represent FXYD2 expression in human renal cortex and panels F and H represent FXYD2 expression in the renal medulla. The expression of the g subunit was less abundant than the a subunit and the distribution of FXYD2 appeared to be limited to the distal nephron (i.e. collecting tubule and the distal convoluted tubule). Original magnification for each panel: 200 (approx.).
(Loffing and Kaissling, 2003). It is plausible that the restricted presence of FXYD2 in the distal nephron may be important for modulating basolateral Na, K-ATPase and the trans-epithelial sodium re-absorption mediated by the concerted action of ENaC and Na, K-ATPase in principal cells. Naturally, it is essential to adapt TMAs for double immunofluorescent labeling (using specific markers of different nephron segments or cell types such as principal cells and intercalated cells) before such a conclusion
can be reached. Therefore the presence of FXYD2 in human kidney is consistent with a major physiological role for the g isoform of Na, K-ATPase in modulating Na, K-ATPase in the renal epithelium (Pu et al., 2001; Farman et al., 2003; PihakaskiMaunsbach et al., 2003). Previous immunocytochemical studies in rat kidney have shown that the FXYD2 and CHIF (FXYD4) are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of
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the Na, K-ATPase activity in the collecting duct (PihakaskiMaunsbach et al., 2003). Our results also confirmed that FXYD2 is expressed in several other tissues from which the cDNA encoding the g protein has been cloned1; i.e. colon, stomach, pancreas, liver and heart (Kim et al., 1997). Expression of FXYD2 in the heart is non-classical and intriguing. Previous studies have used a polyclonal anti-g antiserum to define the tissue specificity of FXYD2 and to determine whether it has a functional role and used western blot analysis to show that the protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells or transfected HeLa cells (Therien et al., 1997). However, the immunohistochemical studies presented here and a recent review of the molecular and functional studies of FXYD2 suggest that it may be expressed in human cardiac muscle (Therien et al., 2001). There may well be differences between the FXYD isoform profiles of human, rodent, canine and porcine cardiac muscle since it is already known that not all Na, K-ATPase isoforms are expressed in human, rodent and guinea pig heart (McDonough et al., 1996). The heart is also a major target of the renin-angiotensinaldosterone system which not only regulates collagen turnover and fibrous tissue formation, but also sodium transport (Delcayre and Swynghedauw, 2002). Until relatively recently it was widely believed that the mineralocorticoid hormone aldosterone (produced in the adrenal cortex) acted exclusively on ion transporting epithelia to promote sodium retention and potassium excretion. However, it is now known that aldosterone also acts on nonepithelial tissues, such as heart and blood vessels (Rocha and Funder, 2002). Therefore expression of FXYD2 in the human heart is perhaps not surprising given the abundance of mineralocorticoid receptors and potential for endocrine regulation of sodium transport. Further experiments are clearly necessary to determine the physiological significance of FXYD2 expression in the human myocardium where three alpha isoforms of Na, K-ATPase are known to be present. FXYD2 protein was also detected in the antral and oxyntic gastric mucosa, in the epididymis, salivary glands, secretory epithelium of the breast and the uterine endometrium. It was also highly abundant in the transport active epithelium of the amnion, placenta and choroid plexus. FXYD2 and FXYD1 were both expressed in the amniotic membrane and placental villi where they may modulate Na, K-ATPase function. In conclusion, the results of this study confirm our hypothesis that the distribution of FXYD1 and FXYD2 proteins in human tissues is more widespread than is apparent from the current literature. These FXYD proteins may associate with Na, K-ATPase in various epithelial and cardiovascular tissues and influence its catalytic properties. Tissue microarray technology will be a useful tool for determining the tissue distribution of novel members of the FXYD gene family as antibodies to these proteins gradually become available.
Acknowledgements This study was funded by the Wellcome Trust UK (Grant number 065559/Z/01/). We express our gratitude to Dr. S.J.D. Karlish (Weizmann Institute of Science, Rehovot, Israel) for providing the polyclonal antibodies to phospholemman and the g subunit of Na, K-ATPase. We are also grateful to Dr. M. Takahashi and Dr. M.W. McEnery for the pan a specific 1 http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/index.html, NCBI AceView, April 2009.
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monoclonal mAb 9A7. We would also like to acknowledge Dr. Cecilia M. Canessa (Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut) and Dr. Nicolette Farman (INSERM Research Unit 478, Faculte´ Xavier Bichat, Paris) for critical comments on the manuscript. References Adams, M.D., Kerlavage, A.R., Fleischmann, R.D., Fuldner, R.A., Bult, C.J., Lee, N.H., Kirkness, E.F., Weinstock, K.G., Gocayne, J.D., White, O., et al., 1995. Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377, 3–174. Arystarkhova, E., Wetzel, R.K., Asinovski, N.K., Sweadner, K.J., 1999. The gamma subunit modulates Na(+ ) and K(+ ) affinity of the renal Na,K-ATPase. J. Biol. Chem. 274, 33183–33185. Attali, B., Latter, H., Rachamim, N., Garty, H., 1995. A corticosteroid-induced gene expressing an ‘‘IsK-like’’ K+ channel activity in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 92, 6092–6096. 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