Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in...

Characterization and regulation of the gene expression of amino acid

transport system A (SNAT2) in rat mammary gland*.

Adriana López 1,2, Nimbe Torres 1, Victor Ortiz 1, Gabriela Alemán 1, Rogelio

Hernández-Pando 3, Armando R. Tovar 1,4.

1 Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias

Médicas y Nutrición “Salvador Zubirán”. México DF, 14000, México; 2 Posgrado en

Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa, México

DF, 09340, México; 3 Departamento de Patología Experimental, Instituto Nacional

de Ciencias Médicas y Nutrición “Salvador Zubirán”. México DF, 14000, México

* This work was supported by The Consejo Nacional de Ciencia y Tecnología

(CONACYT) grant 212226-5-25637M

4 To whom correspondence should be addressed: Armando R. Tovar,

Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas

y Nutrición, Vasco de Quiroga No 15, México, D. F.14000, México. Tel:

(525)6553038. Fax (525)6553038; E-mail: [email protected]

of 35Articles in PresS. Am J Physiol Endocrinol Metab (June 20, 2006). doi:10.1152/ajpendo.00062.2006

Copyright © 2006 by the American Physiological Society.

2

Running Head: System A gene regulation in rat mammary gland

Key words: System A, mammary gland, amino acid transport, lactation

of 35

3

ABSTRACT

Amino acid transport via system A plays an important role during lactation

promoting the uptake of small neutral amino acids, mainly alanine and glutamine.

However, the regulation of gene expression of system A (SNAT2) in mammary

gland has not been studied. The aim of the present work was to understand the

possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation

of gland explants in amino acid-free medium induced the expression of SNAT2,

and this response was repressed by the presence of small neutral amino acids, or

by actinomycin D but not by large neutral or cationic amino acids. The half-life of

SNAT2 mRNA was 67 min indicating a rapid turnover. In addition, SNAT2

expression in the mammary gland was induced by forskolin and PMA, inducers of

PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC

pathways partially prevented the up-regulation of SNAT2 mRNA during adaptive

regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a

lesser extent at peak lactation. β-estradiol stimulated the expression of SNAT 2 in

mammary gland explants, this stimulation was prevented by the estrogen receptor

inhibitor ICI 182780. Our findings clearly demonstrated that the SNAT2 gene is

regulated by multiple pathways, indicating that the expression of this amino acid

transport system is tightly controlled due to its importance for the mammary gland

during pregnancy and lactation to prepare the gland for the transport of amino

acids during lactation.

of 35

4

INTRODUCTION

Milk produced by the mammary gland during lactation is fundamental for the

development and growth of the newborn, providing nutrients in the quantity and

quality required to maintain metabolic functions. Thus, to fulfill this role, the

lactating mammary gland requires, among other nutrients, a large amount of amino

acids to sustain different functions such as synthesis of milk proteins, as energy

substrates (36), and synthesis of fatty acids (39). Several studies in different

species measuring arterio-venous differences in amino acids across the mammary

gland indicate that all amino acids are captured by the gland, with alanine and

glutamine the most actively transported (2, 39). These amino acids are

preferentially transported by system A in most tissues (21), and this activity could

play a key role in the mammary gland as well as in other tissues (3).

Several studies showed that mammary glands possess characteristics of system A

activity (20, 27), as determined by using the non-metabolizable analogue 2-

(methylamino) isobutyrate (MeAIB) (35), such as Na+ dependence, pH sensitivity,

preference for the uptake of short-chain neutral amino acids like alanine and

serine, as well as the amide amino acids glutamine and asparagine, and relatively

lower affinity for threonine and the branched chain amino acids (10, 15, 21). In

addition, system A in mammary gland presents a unique characteristic of this

transport system observed in other cell types called adaptive regulation (35).

Adaptive regulation occurs when cells are incubated in a medium lacking

extracellular amino acid substrates and as a result there is an increase in system A

of 35

5

transport activity. This activity is repressed with actinomycin D, or when cells are

transferred to an amino acid rich medium (14).

In recent years, a gene family of sodium-coupled neutral amino acid transporters

(SNAT; SCL38 gene family) coding for proteins that possess the classically-

described System A transport activities, in terms of their functional properties and

patterns of regulation, has been cloned (18). Several isoforms have been

described and designated SNAT1 (38), SNAT2 or SAT 2 (33, 40), and SNAT4 (34).

SNAT2 is widely expressed in rat tissues, and its mRNA concentration increases in

cultured cells during adaptive regulation or by the addition of cAMP (8). Recent

studies have demonstrated that the increase of SNAT2 expression in response to

amino acid deprivation is associated with the presence of an amino acid response

element (AARE) located in intron 1 of the gene in several species (22).

Due to the importance of system A activity in the mammary gland during lactation,

as well as its role in the control of other amino acid transport systems, it is

important to understand the regulation of expression of the SNAT2 isoform in the

mammary gland. The results of the present study show that SNAT2 expression in

rat mammary gland explants is controlled at multiple levels by a variety of

mechanisms that involve PKA, PKC, as well as estradiol. Presumably, all these

signals tightly control the expression of SNAT2, depending on the physiological

status of the gland during pregnancy and lactation.

of 35

6

MATERIALS AND METHODS

Materials- Nylon membrane filter Hybond-N+, deoxycytidine 5’[α32P] triphosphate

(3000 Ci/mmol) and the rediprime DNA labeling system were from Amersham, UK.

Animals.- Female Wistar rats with a weight of 200-250 g were obtained from the

animal research facility at the Instituto Nacional de Ciencias Médicas y Nutrición.

Animals were housed in individual stainless steel cages at 18oC with a 12-hour

light/dark cycle and allowed free access to water and chow diet. Gestational age

was determined by vaginal smear to detect spermatozoa. Mammary gland explants

were obtained as previously reported (35) from virgin, pregnant, lactating, and

post-weaning rats. After normal pregnancy and delivery, the litter size was

adjusted to 8 pups/dam. Rats at the end of lactation (21 day postpartum) were

separated from their pups. Nonpregnant nonlactacting rats were used as control

rats, and are referred to as “virgin rats”. This study was approved by the Animal

Care Committee of the Instituto Nacional de Ciencias Médicas y Nutrición, México,

in accordance with international guidelines for the use of animals in research.

Preparation of mammary tissue explants. Mammary tissue explants were prepared

as described previously (35). Briefly, rats were anesthetized and then killed by

decapitation. The mammary gland was immediately removed and placed at 37°C in

30 ml Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 95% O2/5% CO2

of 35

7

and contained the following (in mM): 141 NaCl, 5.6 KCl, 3.0 CaCl2, 1.4 KH2PO4,

1.4 MgSO4, 24.6 NaHCO3, and 11 glucose. After removal of the connective tissue,

the mammary tissue was diced into 2- to 5-mg explants. The tissue explants were

rinsed repeatedly with buffer at 37°C prior to assay.

RNA preparation.- Mammary tissue was homogenized in guanidinium buffer

containing 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.1 M 2-

mercaptoethanol, and 0.5% N-laurylsarcosine with a polytron (PT2000,

Kinematica, Switzerland) at the lowest setting. The homogenate was centrifuged at

12,000 X g for 15 minutes at 18°C, and the resulting supernatant was layered onto

a cesium chloride solution containing 5.7 M CsCl and 25 mM sodium acetate, pH

5.2. The cesium chloride gradient was formed by centrifugation at 113,000 X g for

18 hours at 18°C to yield total RNA. The RNA was precipitated with 100% ethanol

and 3M sodium acetate, pH 5.2, washed twice in 75% ethanol and resuspended in

formamide. The RNA was quantified by optical density at 260 nm, and stored at -

80°C until use.

Probe preparation- The probe was prepared by RT-PCR using poly(A)+ mRNA

isolated from mammary gland. Poly(A)+ RNA was purified from mammary gland

explants by guanidine thiocyanate extraction and ultracentrifugation through a

cesium chloride cushion (5) followed by a single round of oligo(dT) cellulose

chromatography. The sense primer was 5’-CTACTCATACCCCACGAAGCA-3’

which corresponds to the nucleotide positions 475-495 in SNAT2 cDNA and the

of 35

8

antisense primer was 5’ ACGACTACGCCACTCAGGA-3’, which corresponded to

nucleotide positions 1824-1842 in SNAT2 cDNA (GenBank TM accession number

NM181090). RT-PCR was performed using the following PCR amplification

conditions: denaturation for 5 min at 95°C; annealing for 1 min at 56.2 °C and

extension for 1.30 min at 72°C for 34 cycles; final extension for 7 min at 72°C. The

resultant product (1368 bp) was separated by electrophoresis on a 1.5 %

agarose/Tris/acetate/EDTA gel containing ethidium bromide and viewed under UV

light. Afterward, manual sequencing of the product was performed to establish its

molecular identity.

Northern blot analysis. For Northern analysis, 15 micrograms of RNA were loaded

into each lane and were electrophoresed in a 1.0% agarose gel containing 37%

formaldehyde. The RNA was then blotted onto nylon membranes (Hybond-N+) and

cross-linked with a UV crosslinker (Amersham). cDNA probe was labeled with

Redivue [α -32P]dCTP (110 TBq/mmol) using the Rediprime DNA labeling kit.

Membranes were prehybridized with rapid-hyb buffer (Amersham) at 65°C for 1

hour, and then hybridized with the cDNA probe for 2.5 hours at 65°C. Membranes

were washed once with 2 X SSC/0.1% SDS (1 X SSC = 0.15 M sodium chloride,

15 M sodium citrate) at room temperature for 20 minutes and then twice for 15 min

with 0.1 X SSC/0.1% SDS at 65°C. Digitization of the images and quantitation of

radioactivity (cpm) of the bands were done by using the Instant Imager (Packard

Instrument, Meriden, CT). Membranes were also exposed to Extascan film (Kodak,

Mexico) at -70°C with an intensifying screen.

of 35

9

Preparation of tissue for in situ RT-PCR. The alcohol-fixed mammary gland

sections (4 µm) were embedded in paraffin and used for system A mRNA

expression determined by in situ RT-PCR. Mammary gland sections were mounted

on silane cover slides, deparaffinized for 18 hr at 60°C and sequentially immersed

in xylene (30 min at 37°C), absolute ethanol, 50% ethanol and water. Cells were

rendered permeable by incubation for 10 min at room temperature in 0.02% HCl,

followed by a 90-second incubation with 0.01% Triton-X-100 and then a 30-min

incubation at 37°C with 1 µg/ml of proteinase K (Gibco BRL, Gaithersburgh, MD).

DNA was removed by incubation for 15 min at 37°C with DNase, 3U per tissue

section (Gibco BRL). Reverse transcription was performed at 37°C for 1 hr,

incubating the tissue in 50 µl of reverse transcriptase buffer (1X, Gibco BRL), 0.1 M

dithiothreitol (DTT), 2 mM dNTP mix (Gibco BRL), 0.1 µg/µl of oligo dT, and 200 U

of Moloney murine leukaemia virus reverse transcriptase (Gibco BRL). The

sections were sealed using the Assembly tool (Perkin-Elmer, Branchburg, NJ). The

RNA was then removed by incubation with 3 U of RNase and fixed again by

immersion with 4% paraformaldehyde in Sorensen buffer for 3 min at 4°C, PCR

was performed, incubating the tissue sections with 50 µl of 1X reaction buffer

(Gibco BRL), 2 U of Taq polymerase, 15 mM MgCl2 and dNTPs labeled with

digoxigenin (Boehringer Mannheim, Mannheim, Germany). The slides were sealed

again and placed in the thermocycler (Perkin Elmer). After an initial denaturation at

95°C for 5 min, 35 cycles were performed using as annealing temperature of

56.2°C for 1 min and as extension temperature of 72°C for 1.5 min. PCR products

were detected with monoclonal mouse antidigoxigenin antibodies coupled to

of 35

10

alkaline phosphatase and tetrazolium nitroblue (Boehringer Mannheim), and

counterstained using nuclear fast red. Expression of actin was used as a positive

control, and the negative control consisted of performing the whole procedure

without the system A primers.

RESULTS

To determine the mechanism(s) of regulation of the SNAT2 gene expression, we

studied whether adaptive regulation was accompanied with a concomitant increase

of SNAT2 mRNA in mammary gland explants. With this purpose, explants were

incubated in an amino acid-free medium for different periods of time up to 300 min.

As shown in Fig. 1, SNAT2 mRNA concentration increased steadily during the first

250 min of amino acid deprivation, reaching a 20-fold induction. When incubation

of mammary gland explants in amino acid-free medium was prolonged up to 300

min, SNAT2 mRNA concentration began to decay. Interestingly, when mammary

gland explants were incubated with an equimolar mixture of the amino acids used

by the amino acid transport system A (alanine, glycine and serine 1mM each), the

induction of SNAT2 was suppressed (Fig 1). However, when explants were

incubated with a mixture of large neutral and cationic amino acids (phenylalanine,

valine and lysine 1mM each), a rapid induction of SNAT2 was observed at minute

50, and it was less prolonged in comparison with explants incubated in an amino

acid-free medium (Fig 1). These results clearly indicated that SNAT2 gene

expression in mammary gland explants was susceptible to the presence or

absence of small neutral amino acids in the medium. To identify if the expression

of 35

11

of SNAT2 was dependent on RNA synthesis, lactating mammary gland explants

were preincubated for 300 min in the presence of 50 µM actinomycin D. As can be

seen in Fig 1 there was no induction of SNAT2 mRNA indicating that abundance of

SNAT2 mRNA was indeed dependent on RNA synthesis during adaptive

regulation.

To study the rate of SNAT2 mRNA decay, explants were preincubated for 200 min

in an amino acid-free medium, and were then switched to a medium containing a

mixture of small neutral amino acids (alanine, glycine, and serine 1mM each) to

repress SNAT2 gene expression. A rapid decay of SNAT2 mRNA was observed,

and the estimated half-life for the decay was 67 min, indicating that SNAT2 mRNA

has a rapid turnover rate. These results indicate that the presence of the amino

acid substrates for system A in the medium rapidly repressed the expression of

SNAT2 mRNA. (Fig 2).

It has been demonstrated in various tissues that system A activity is up-regulated

by several hormones (32). Glucagon as well as dibutyril-cAMP significantly

stimulate system A activity and SNAT2 gene expression in liver cells (15).

However, there is evidence that rat mammary gland lacks of glucagon receptor

(37). We then studied whether SNAT2 mRNA expression in mammary gland

explants was responsive to distinct signaling pathways, one coupled to

phospholipase C, generating diacylglycerol and inositol 1,4,5-triphosphate, and the

other coupled to adenylate cyclase, increasing cAMP levels. To assess if any of

these signaling pathways were involved in regulating system A gene expression in

of 35

12

mammary gland, explants were incubated in medium containing alanine, glycine

and serine at 1 mM each, in the presence of 10 µM forskolin or 100 nM PMA. The

results showed a strong induction with either compound, indicating that signaling

can occur via PKA or PKC (Fig 3).

To determine whether stimulation of SNAT2 gene expression during adaptive

regulation was mediated via PKA or PKC, mammary gland explants were

incubated with the specific inhibitors of PKA or PKC, H89 or GF109203X,

respectively, and the inhibitors for adenylate cyclase or phospholipase C, MDL

12330 HCl or ET-18-CH3, respectively, in an amino acid free-medium for 200 min

(Fig 4). As expected, explants incubated in the amino acid free-medium showed an

increase in SNAT2 mRNA expression by approximately 5-fold, however, the

presence of any of the inhibitors prevented the induction of the SNAT2 gene

indicating that inhibition of both signaling pathways reduced SNAT2 gene

expression. As expected, incubation of mammary gland explants in an amino acid-

free medium in the presence of forskolin or PMA increased SNAT2 mRNA

concentration in comparison with explants incubated with a medium containing

amino acids (Fig 4).

These results indicated that SNAT2 expression is regulated by different signal

transduction pathways in lactating mammary gland, however, it has not been

established if the SNAT2 gene is expressed differentially during pregnancy and

lactation. Thus, experiments were designed to determine if the expression of

SNAT2 mRNA changed during pregnancy or lactation. Mammary gland explants

of 35

13

from rats at different stages of pregnancy, lactation and weaning were studied.

Northern blot analysis revealed a single band of 4.5 kb corresponding to SNAT2

mRNA in explants of mammary gland derived from pregnant and lactating rats.

Furthermore, SNAT2 mRNA concentration increased progressively during

pregnancy until day 18, and decreased rapidly near the end of pregnancy. During

lactation the pattern of expression of SNAT2 mRNA showed small fluctuation

without considerable changes in the first days of lactation. However, there was an

increase of SNAT2 mRNA concentration around day 12 to 16 that coincided with

peak milk production (Fig 5). After postweaning SNAT2 mRNA decreased rapidly.

This finding was strengthened by in situ RT-PCR in mammary gland explants,

indicating that lactocytes increased SNAT2 mRNA concentration predominantly

during pregnancy (Fig 6). To understand if SNAT2 expression was induced by

hormonal changes occurring during pregnancy, we performed studies in lactating

mammary gland explants incubated in the presence of progesterone, 17β-estradiol,

or both. The addition of progesterone to explants incubated in a medium containing

alanine, glycine and serine at 1 mM each to repress adaptive regulation, did not

change the expression of SNAT2 mRNA. However, the incubation of explants in

the same medium in the presence of 17β-estradiol markedly increased the

concentration of SNAT2 mRNA. The effect of 17β-estradiol was time-dependent

and after 3h of incubation SNAT2 mRNA increased by almost 6-fold (Fig 7). The

induction of SNAT2 with 17β estradiol was repressed by the addition of the specific

inhibitor for estrogen receptor alpha, ICI 182,780 M (Fig. 8). These results indicate

of 35

14

an additional level of regulation of SNAT2 gene expression involving hormones

present during pregnancy.

DISCUSSION

The results of this study demonstrated that expression of SNAT2 in the mammary

gland is regulated by different effectors. As expected, incubation of mammary

gland explants in an amino acid-free medium showed adaptive regulation. These

results are in agreement with previous reports that show that the activity of system

A, measured as uptake of MeAIB into mammary gland explants, increases if tissue

is incubated in an amino acid free medium (35). The increase in SNAT2 mRNA

during adaptive regulation in the mammary gland explants follow similar kinetics to

those observed in HepG2 cells or fibroblasts (1, 4, 17). Expression of SNAT2

mRNA occurs rapidly in HepG2 cells after incubating the explants in the amino

acid-free medium (1). The expression was sensitive to the presence of amino

acids, mainly the small neutral amino acids, since the mixture of alanine, glycine

and serine prevented the elevation of SNAT2 mRNA concentration, but this

reduction was not observed with a mixture of large neutral and cationic amino

acids. Adaptive regulation of SNAT2 gene is controlled at the transcriptional level,

studies by Kilberg and coworkers have determined that a specific amino acid

response element is located in the intron 1 and is responsible for regulating SNAT2

expression under conditions of amino acid deprivation (22).

of 35

15

The estimated half-life of SNAT2 mRNA was approximately 67 min, indicating the

rapid degradation rate of SNAT2 mRNA. This short half-life is in agreement with

others estimated by measuring the activity of system A after treatment with

glucagon that was between 1.4 and 1.5 h for both in vivo and in vitro studies (6).

This rapid turnover allows for tight regulation of utilization of amino acids such as

alanine and glutamine, the amino acids more actively transported in the mammary

gland (2, 39). Nonetheless, is not clear what is the physiological relevance of

adaptive regulation in the mammary gland, since there is evidence that despite

dams consume a low or an adequate protein diet, the intracellular pool during the

peak of lactation of amino acids including the amino acid substrates for system A is

not depleted (11).

In addition to adaptive regulation, several studies have demonstrated that system

A activity is induced by glucagon in different cells and tissues (15). Recent

evidence demonstrated that cAMP also induces the expression of SNAT2 in

cultured cells (8), and these changes have been associated with an increase in

system A activity. We previously demonstrated that the activity of this transport

system is also increased by dibutyril cAMP in the mammary gland (35). The

present study clearly demonstrated that the increase of system A transport activity

by cAMP is mediated, in part, by an increase in the expression of SNAT2 mRNA.

Our results also showed that the expression of this gene is activated by adenylate

cyclase and phospholipase C-dependent pathways (7). As a result of the

activation via both pathways, it is not clear if SNAT2 gene expression is

preferentially activated via PKA or PKC. Surprisingly, our data show that SNAT2

of 35

16

expression is up-regulated by both signal transduction pathways. The induction

with either pathway was similar, indicating that transcription of SNAT2 is activated

in the same fashion via PKA or PKC. There is evidence that during pregnancy,

cAMP levels increase in rodent mammary gland, and then fall rapidly after

parturation (23), and perhaps this explains, in part, the increase in expression of

SNAT2 during pregnancy, although there are other factors that influence the

expression of this gene as described below.

Studies measuring amino acid arterio-venous differences concentrations across

the mammary gland show that uptake of most amino acids including the amino

acids transported by system A occurs very actively during lactation (26, 28).

However, the pattern of expression of SNAT2 in the mammary gland during

pregnancy and lactation has not been determined previously. Our data show that

up-regulation of SNAT gene expression occurs mainly during pregnancy and

during the peak of lactation. These results suggest that SNAT2 gene expression is

probably regulated during pregnancy by hormonal changes that occur in this

condition. Our data show that SNAT2 gene expression is induced by β-estradiol,

but not by progesterone. This is in agreement with previous studies that show an

increase in the activity of system L and A in breast cancer cell lines incubated with

β-estradiol (29, 30). Induction of system A by estrogens in breast cancer cell lines

only occurred in those expressing estrogen receptor-α . During pregnancy, it has

been shown that expression of estrogen receptor-α decreases, but estrogen

receptor-β remains high (24, 25), however, there is no experimental evidence that

of 35

17

associates expression of SNAT2 with the presence of this receptor. More research

is needed to understand this interaction. In addition, due to the rapid induction of

SNAT2 by β-estradiol in the mammary gland explants, there is the possibility that

this effect could be mediated by a non genomic action of the estrogen receptor (13,

19). It has been demonstrated that estrogen receptor-α is also present in the

plasma membrane, and that binding of β-estradiol activates cellular signaling

systems that involve either Gαs and Gαq proteins (19). Furthermore, it has been

shown that β-estradiol is able to activate by a non genomic mechanism

phospholipase C at the membrane and stimulates PKC pathway in osteoblasts

(16), and also activates PKA and PKC in neurons altering synaptic transmissions in

the cells (12, 31). Further studies are required to understand whether activation of

SNTA2 by β-estradiol occurs through a genomic or non genomic mechanism.

It is not clear why expression of SNAT2 increases during pregnancy and

decreases during the first part of lactation. Amino acids are needed for lactocyte

formation during pregnancy, but are also needed for synthesis of milk lipids and

proteins during lactation. This change in expression could also be associated with

the possibility that the mammary gland is preparing the milk-producing cells for

lactation by synthesizing amino acid transporters during pregnancy for lactation. It

has been shown that system A protein in skeletal muscle is located in vesicles that

can recycle to the plasma membrane to increase activity in response to insulin (9).

Perhaps the synthesis of system A protein is increased and that located in

intracellular vesicles is cycled to the membrane after receiving a hormonal signal

of 35

18

that occurs during lactation. We are conducting studies to detect if in fact this event

takes place.

of 35

19

REFERENCES

1. Bain PJ, LeBlanc-Chaffin R, Chen H, Palii SS, Leach KM, and Kilberg

MS. The mechanism for transcriptional activation of the human ATA2

transporter gene by amino acid deprivation is different than that for

asparagine synthetase. J Nutr 132: 3023-3029, 2002.

2. Barber T, Triguero A, Martinez-Lopez I, Torres L, Garcia C, Miralles VJ,

and Vina JR. Elevated expression of liver gamma-cystathionase is required

for the maintenance of lactation in rats. J Nutr 129: 928-933, 1999.

3. Bussolati O, Dall'Asta V, Franchi-Gazzola R, Sala R, Rotoli BM, Visigalli

R, Casado J, Lopez-Fontanals M, Pastor-Anglada M, and Gazzola GC.

The role of system A for neutral amino acid transport in the regulation of cell

volume. Mol Membr Biol 18: 27-38, 2001.

4. Gazzola RF, Sala R, Bussolati O, Visigalli R, Dall'Asta V, Ganapathy V,

and Gazzola GC. The adaptive regulation of amino acid transport system A

is associated to changes in ATA2 expression. FEBS Lett 490: 11-14, 2001.

5. Glisin V, Crkvenjakov R, and Byus C. Ribonucleic acid isolated by cesium

chloride centrifugation. Biochemistry 13: 2633-2637, 1974.

6. Handlogten ME and Kilberg MS. Induction and decay of amino acid

transport in the liver. Turnover of transport activity in isolated hepatocytes

after stimulation by diabetes or glucagon. J Biol Chem 259: 3519-3525,

1984.

of 35

20

7. Hansen LH, Gromada J, Bouchelouche P, Whitmore T, Jelinek L,

Kindsvogel W, and Nishimura E. Glucagon-mediated Ca2+ signaling in

BHK cells expressing cloned human glucagon receptors. Am J Physiol 274:

C1552-1562, 1998.

8. Hatanaka T, Huang W, Martindale RG, and Ganapathy V. Differential

influence of cAMP on the expression of the three subtypes (ATA1, ATA2,

and ATA3) of the amino acid transport system A. FEBS Lett 505: 317-320,

2001.

9. Hyde R, Peyrollier K, and Hundal HS. Insulin promotes the cell surface

recruitment of the SAT2/ATA2 system A amino acid transporter from an

endosomal compartment in skeletal muscle cells. J Biol Chem 277: 13628-

13634, 2002.

10. Hyde R, Taylor PM, and Hundal HS. Amino acid transporters: roles in

amino acid sensing and signalling in animal cells. Biochem J 373: 1-18,

2003.

11. Jansen GR, Schibly MB, Masor M, Sampson DA, and Longenecker JB.

Free amino acid levels during lactation in rats: effects of protein quality and

protein quantity. J Nutr 116: 376-387, 1986.

12. Kelly MJ, Lagrange AH, Wagner EJ, and Ronnekleiv OK. Rapid effects of

estrogen to modulate G protein-coupled receptors via activation of protein

kinase A and protein kinase C pathways. Steroids 64: 64-75, 1999.

of 35

21

13. Kelly MJ and Levin ER. Rapid actions of plasma membrane estrogen

receptors. Trends Endocrinol Metab 12: 152-156, 2001.

14. Kilberg MS, Pan YX, Chen H, and Leung-Pineda V. Nutritional control of

gene expression: how mammalian cells respond to amino acid limitation.

Annu Rev Nutr 25: 59-85, 2005.

15. Kilberg MS, Stevens BR, and Novak DA. Recent advances in mammalian

amino acid transport. Annu Rev Nutr 13: 137-165, 1993.

16. Le Mellay V, Grosse B, and Lieberherr M. Phospholipase C beta and

membrane action of calcitriol and estradiol. J Biol Chem 272: 11902-11907,

1997.

17. Ling R, Bridges CC, Sugawara M, Fujita T, Leibach FH, Prasad PD, and

Ganapathy V. Involvement of transporter recruitment as well as gene

expression in the substrate-induced adaptive regulation of amino acid

transport system A. Biochim Biophys Acta 1512: 15-21, 2001.

18. Mackenzie B and Erickson JD. Sodium-coupled neutral amino acid

(System N/A) transporters of the SLC38 gene family. Pflugers Arch 447:

784-795, 2004.

19. Moggs JG and Orphanides G. Estrogen receptors: orchestrators of

pleiotropic cellular responses. EMBO Rep 2: 775-781, 2001.

20. Neville MC, Lobitz CJ, Ripoll EA, and Tinney C. The sites for alpha-

aminoisobutyric acid uptake in normal mammary gland and ascites tumor

of 35

22

cells. A comparative study of mouse tissues in vitro. J Biol Chem 255: 7311-

7316, 1980.

21. Palacin M, Estevez R, Bertran J, and Zorzano A. Molecular biology of

mammalian plasma membrane amino acid transporters. Physiol Rev 78:

969-1054, 1998.

22. Palii SS, Chen H, and Kilberg MS. Transcriptional control of the human

sodium-coupled neutral amino acid transporter system A gene by amino

acid availability is mediated by an intronic element. J Biol Chem 279: 3463-

3471, 2004.

23. Rillema JA. Cyclic nucleotides, adenylate cyclase, and cyclic AMP

phosphodiesterase in mammary glands from pregnant and lactating mice.

Proc Soc Exp Biol Med 151: 748-751, 1976.

24. Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, and Gustafsson

JA. Estrogen receptors alpha and beta in the rodent mammary gland. Proc

Natl Acad Sci U S A 97: 337-342, 2000.

25. Saji S, Sakaguchi H, Andersson S, Warner M, and Gustafsson J.

Quantitative analysis of estrogen receptor proteins in rat mammary gland.

Endocrinology 142: 3177-3186, 2001.

26. Shennan DB. Mammary gland membrane transport systems. J Mammary

Gland Biol Neoplasia 3: 247-258, 1998.

of 35

23

27. Shennan DB and McNeillie SA. Characteristics of alpha-aminoisobutyric

acid transport by lactating rat mammary gland. J Dairy Res 61: 9-19, 1994.

28. Shennan DB, Millar ID, and Calvert DT. Mammary-tissue amino acid

transport systems. Proc Nutr Soc 56: 177-191, 1997.

29. Shennan DB, Thomson J, Barber MC, and Travers MT. Functional and

molecular characteristics of system L in human breast cancer cells. Biochim

Biophys Acta 1611: 81-90, 2003.

30. Shennan DB, Thomson J, Gow IF, Travers MT, and Barber MC. L-

leucine transport in human breast cancer cells (MCF-7 and MDA-MB-231):

kinetics, regulation by estrogen and molecular identity of the transporter.

Biochim Biophys Acta 1664: 206-216, 2004.

31. Shingo AS and Kito S. Estradiol induces PKA activation through the

putative membrane receptor in the living hippocampal neuron. J Neural

Transm 112: 1469-1473, 2005.

32. Shotwell MA, Kilberg MS, and Oxender DL. The regulation of neutral

amino acid transport in mammalian cells. Biochim Biophys Acta 737: 267-

284, 1983.

33. Sugawara M, Nakanishi T, Fei YJ, Huang W, Ganapathy ME, Leibach

FH, and Ganapathy V. Cloning of an amino acid transporter with functional

characteristics and tissue expression pattern identical to that of system A. J

Biol Chem 275: 16473-16477, 2000.

of 35

24

34. Sugawara M, Nakanishi T, Fei YJ, Martindale RG, Ganapathy ME,

Leibach FH, and Ganapathy V. Structure and function of ATA3, a new

subtype of amino acid transport system A, primarily expressed in the liver

and skeletal muscle. Biochim Biophys Acta 1509: 7-13, 2000.

35. Tovar AR, Avila E, DeSantiago S, and Torres N. Characterization of

methylaminoisobutyric acid transport by system A in rat mammary gland.

Metabolism 49: 873-879, 2000.

36. Tovar AR, Becerril E, Hernandez-Pando R, Lopez G, Suryawan A,

Desantiago S, Hutson SM, and Torres N. Localization and expression of

BCAT during pregnancy and lactation in the rat mammary gland. Am J

Physiol Endocrinol Metab 280: E480-488, 2001.

37. Trottier NL. Nutritional control of amino acid supply to the mammary gland

during lactation in the pig. Proc Nutr Soc 56: 581-591, 1997.

38. Varoqui H, Zhu H, Yao D, Ming H, and Erickson JD. Cloning and

functional identification of a neuronal glutamine transporter. J Biol Chem

275: 4049-4054, 2000.

39. Vina JR and Williamson DH. Effects of lactation on L-leucine metabolism

in the rat. Studies in vivo and in vitro. Biochem J 194: 941-947, 1981.

40. Yao D, Mackenzie B, Ming H, Varoqui H, Zhu H, Hediger MA, and

Erickson JD. A novel system A isoform mediating Na+/neutral amino acid

cotransport. J Biol Chem 275: 22790-22797, 2000.

of 35

25

FIGURE LEGENDS

Figure 1. Expression of SNAT2 mRNA in mammary gland explants from lactating

rats incubated in (Panel A) an amino acid-free medium up to 300 min, or in

medium containing a mixture of small neutral (ala, gly, and ser), or large neutral

and cationic amino acids (phe, val, lys), or in an amino acid-free medium

containing actinomycin D. Panel B shows the quantitative analysis of mRNA

concentration of the experiments indicated in panel A. Values were normalized with

ribosomal RNA 28S. Values are mean ± SEM (n = 3).

Figure 2. Rate of decay of mammary gland SNAT2 mRNA from rat explants after

200 min of preincubation in an amino acid-free medium, and then incubated up to 2

h in a medium containing a mixture of small neutral amino acids (ala, gly and ser)

(Panel A). Quantitative analysis of mRNA concentration is shown in panel B.

Values are mean ± SEM (n = 3).

Figure 3. Effect of 10 µM forskolin or 100 nM PMA on SNAT2 mRNA expression on

rat mammary gland explants incubated in a medium containing a mixture of small

neutral amino acids (ala, gly and ser, 1 mM), panel A. Panel B shows the

quantitative analysis of SNAT2 mRNA. Values are mean ± SEM (n = 3).

Figure 4. Effect of specific inhibitors of protein kinases A or C, and adenylate

cyclase and phospholipase C on the expression of SNAT2 mRNA in rat mammary

of 35

26

gland explants incubated an amino acid-free medium (Panel A). Panel B shows the

quantitative analysis of SNAT2 mRNA. Values are mean ± SEM (n = 3).

Figure 5. Northern blot analysis of SNAT2. Panel A shows the pattern of

expression of SNAT2 mRNA in rat mammary gland during pregnancy, lactation

and postweaning. Panel B shows the abundance of SNAT2 mRNA estimated by

use of the Instant Imager electronic autoradiography system. Values are mean ±

SEM (n = 3).

Figure 6. In situ RT-PCR of SNAT2 mRNA expression in virgin (x200), pregnant

(x100), lactating (x100) and postweaning (x100) rat mammary gland explants.

SNAT2 mRNA was amplified after reverse transcription with the primers indicated

in the Material and Methods section .

Figure 7. Effect of 17β-estradiol (panel A) or progesterone (panel C) in rat

mammary gland explants incubated in a medium containing a mixture of small

neutral amino acids (ala, gly, ser, 1mM each). Panel B and D show the quantitative

analysis of SNAT2 mRNA. Values are mean ± SEM (n = 3); different letters

indicate a significant difference among groups (P< 0.05).

Figure 8. Inhibition of induction of SNAT2 mRNA concentration by 17β estradiol

with the specific inhibitor for estrogen receptor-α, ICI 182,780M (Panel A). Panel B

shows the concentration of SNAT2 mRNA estimated by use of the Instant Imager

of 35

27

electronic autoradiography system. Values are mean ± SEM (n = 3); different

letters indicate a significant difference among groups (P< 0.05).

of 35

A

B

0

5

10

15

20

25

30

0 50 100 150 200 250 300 350

Free-amino acid medium

ala, gly, ser 1mM each

phe, val, lys 1 mM each

Actinomycin D

Rel

ativ

e to

bas

al

Time (min)

basal

Fig 1

of 35

A

B

Fig 2

0

1

2

3

4

5

6

7

0 20 40 60 80 100 120 140

1 mM Ala, Gly, SerNo preincubation

Rel

ativ

e ab

un

dan

ce(%

of

bas

al)

Incubation time (min)

of 35

0

5

10

15

20

25

30

35

0 50 100 150 200 250 300 350

ForskolinPMA

Rel

ativ

e to

bas

al

Time (min)

A

B

Fig 3

of 35

0

1

2

3

4

5

6

+ AA

- AA

H89

MDL-12330A HCl

forskolin

GF 109203X

ET-18-CH3

PMA

Rel

ativ

e un

its

A

B

Fig 4

of 35

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Rel

ativ

e to

bas

al

Time (d)

Pregnancy Lactation Post-weaning

A

B

Fig 5

of 35

Virgin Gestation

Lactation Postweaning

Fig 6

of 35

Fig 7

0

1

2

3

4

5

6

7

0 1 2 3

Rel

ativ

e to

bas

al

Time (h)

0

1

2

3

4

5

6

7

0 1 2 3R

elat

ive

to b

asal

Time (h)

A

B

C

D

a

b

c

d

aa,b a,b

b

of 35

0

2

4

6

8

basal -AA +AA +AA+E2

10-6 10-7 10-8

Rel

ativ

e to

bas

al

E2 + ICI 182,780M

(M)

a

b

cc

cc

c

A

B

Fig 8

of 35

Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in...

Documents

Transcript of Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in...