Acute heat stress and thermal acclimation induce CCAAT/enhancer-binding protein delta in the goby...

ORIGINAL PAPER

Acute heat stress and thermal acclimation induce CCAAT/enhancer-binding protein delta in the goby Gillichthys mirabilis

Bradley A. Buckley

Received: 12 November 2010 / Revised: 28 February 2011 / Accepted: 12 March 2011 / Published online: 27 March 2011

� Springer-Verlag 2011

Abstract Members of the CCAAT/enhancer-binding

protein (C/EBP) family of transcription factors have reg-

ulatory control over numerous processes related to cell fate

determination, including differentiation, proliferation, cell

cycle arrest and apoptosis. In mammals, abnormalities in

the expression of some isoforms of C/EBPs are pathogenic

and are implicated as being involved in myeloid leukemia

and breast cancers. Next to nothing is known about their

regulation, function or stress-responsiveness in poikilo-

therms. Here, both acute heat stress and thermal acclima-

tion were demonstrated to induce the expression of one

isoform, C/EBP-d, in the liver, white muscle and gill of the

eurythermal estuarine goby, Gillichthys mirabilis. The

established role of C/EBP-d in causing cell cycle arrest

and/or promoting apoptosis in other vertebrates suggests

that the heat-inducibility of this protein in poikilotherms

may be part of the conserved cellular stress response with

the hypothesized role of causing temporary cessation of

cell growth and/or programmed cell death during bouts of

environmental stress. The observed regulation of c/ebp-dduring hyperthermia represents a novel, heat-inducible

signaling pathway in fishes.

Keywords Heat shock � Fishes � Cellular stress response �Transcriptional regulation

Introduction

The CCAAT/enhancer-binding protein (C/EBP) family of

transcription factors is a group of basic leucine zipper

(bZIP) proteins that are involved in regulating numerous

biological processes including cell cycle progression, cell

growth and differentiation, apoptosis, and immune and

inflammatory responses (Wedel and Zeigler-Heitbrock

1995; Johnson 2005; Nerlov 2007). In mammals, the

family consists of six known isoforms (designated C/EBP-

a, -b, -c, -d, -e, and -f) whose functions differ depending on

cell- and tissue-type (Ramji and Foka 2002). In response to

isoform-specific cues, such as the application of inflam-

matory cytokines or the removal of essential growth fac-

tors, all isoforms of C/EBP are phosphorylated, form

dimers, translocate to the nucleus and bind to a consensus

ATTGCGCAAT domain in the promoters of their target

genes (Johnson 1993; Osada et al. 1996; Ramji and Foka

2002; Miller et al. 2003). The expression patterns of the

C/EBPs have been linked to changes in cell fate (Nerlov

2007), as some isoforms drive the maturation of given cell

types such as hematopoietic cells down specific develop-

mental trajectories (Rosenbauer and Tenen 2007; Gery and

Koeffler 2009). In other contexts members of this family of

transcription factors have the ability to regulate cell growth

arrest and apoptosis (O’Rourke et al. 1999; Thangaraju

et al. 2005).

As the expression of one C/EBP isoform, C/EBP-d, has

been determined to be abnormal in pathological states

including human breast cancers and acute myeloid leuke-

mia (Porter et al. 2003; Milde-Langosch et al. 2003; Tang

Communicated by H.V. Carey.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00360-011-0572-4) contains supplementarymaterial, which is available to authorized users.

B. A. Buckley (&)

Department of Biology, Portland State University,

Portland, OR 97201, USA

e-mail: bbuckley@pdx.edu

123

J Comp Physiol B (2011) 181:773–780

DOI 10.1007/s00360-011-0572-4

et al. 2006; Gery and Koeffler 2009), considerable attention

has been focused on its structure, function, regulation and

target genes in mammalian cell lines (Sivko and DeWille

2004; Sivko et al. 2004; Gery et al. 2005; Zhang et al.

2008; Gery and Koeffler 2009). There is a growing

understanding that C/EBP-d has the potential to act, in

some cellular contexts, as a cell cycle ‘‘stop sign’’ and that

abnormalities in its expression may lead to inappropriate

cellular proliferation (Lekstrom-Himes and Xanthopolous

1998; Johnson 2005). Interestingly, in studies on molecular

markers of tumor formation in human breast tissue, c/ebp-dwas one of only 34 genes that were under-expressed in

tumor cells, perhaps contributing to their pathogenically

unchecked division (Porter et al. 2001, 2003). It has also

been shown, however, that in mammary epithelial cells,

C/EBP-d induces the expression of the pro-apoptotic genes

p53, BAK, and IGFBP5 and represses the anti-apoptotic

genes encoding BFL1 and Cyclin D1 (Thangaraju et al.

2005). Thus, it appears that C/EBP-d induction may in

some cases promote programmed cell death.

Patterns of c/ebp-d expression during development have

been described in the zebrafish, Danio rerio (Lyons et al.

2001). To date, however, little is known about the function

of this transcription factor in poikilotherms or about its

responsiveness to environmental temperature. In an earlier

transcriptional profiling study, c/ebp-d was one of hundreds

of genes that were induced by acute heat stress in the gill

and white muscle of the current study species, the eury-

thermal estuarine goby Gillichthys mirabilis (Buckley et al.

2006). Whether the observed heat-induction of c/ebp-d at

the mRNA level is linked to cell cycle arrest, apoptosis

or other cellular processes implicated by studies in mam-

malian cell lines is unknown. Furthermore, changes in

c/ebp-d mRNA levels do not necessarily predict changes at

the protein level and, as the production and activity of

C/EBP-d in mammalian tissues is subject to both post-

transcriptional and post-translational regulation (Dearth

and DeWille 2003; Sivko et al. 2004), a protein-level

analysis is required to begin to explore the functional sig-

nificance of the heat-responsiveness of this transcription

factor in fishes.

The specific goals of the current study were: (1) to

establish the kinetics and tissue-specificity of C/EBP-dproduction at the protein level in response to acute heat

stress, (2) to determine the effect of longer-term thermal

acclimation on C/EBP-d levels, and (3) to begin to explore

the regulatory pathways governing C/EBP-d expression in

fishes by screening a region upstream from the G. mirabilis

c/ebp-d start codon for binding sites of known regulators of

C/EBP-d expression in other vertebrates. The findings

support the hypothesis that C/EBP-d may act as a novel

member of the suite of transcription factors responsible for

coordinating the cellular stress response.

Materials and methods

Animal collection

Specimens of the longjaw mudsucker G. mirabilis

(Cooper) were collected by baited minnow trap from the

lagoon on the campus of the University of California at

Santa Barbara, CA, USA (3402400N, -1190420W00). Fish

were transported to Stanford University’s Hopkins Marine

Station in aerated coolers and maintained in flowing sea-

water aquaria for 4 weeks. Fish were exposed during this

period to ambient seawater temperatures (16 ± 1.5�C). For

the first 3 weeks of laboratory acclimation, fish were fed

trout pellets (Bio-Oregon, Warrenton, OR, USA) every

other day.

Acute heat treatment

Fish were held in a 400-l, re-circulating seawater aquarium,

under constant aeration. The tank was ramped from 18 to

32�C at a rate of 0.08�C/min. This heating rate is similar to

that experienced during the course of a day in this species’

natural habitat (Buckley and Hofmann 2002). N = 4 indi-

viduals were removed at 0, 180, 240, 270, 300, 360, and

480 min after initiation of the heat treatment and sacrificed

via cervical transection. Gill, white muscle and liver tissues

were removed and flash frozen in liquid N2.

Thermal acclimation treatment

Fish were held in 400-l, re-circulating seawater aquaria,

under constant aeration for 30 days at either 15�C (n = 5)

or 25�C (n = 5). Fish were fed trout pellets every other day

during this period. After the acclimation period, fish were

sacrificed via cranial concussion followed by cervical

transection. Following sacrifice, gill, white muscle and

liver tissues were removed and flash frozen in liquid N2.

Cloning of the G. mirabilis c/ebp-d cDNA

A 641-bp fragment of the c/ebp-d cDNA was cloned from

G. mirabilis as part of the construction of an expressed

sequence tag (EST) microarray from this species that was

used in previous studies (for complete descriptions of the

construction of the G. mirabilis microarray see Gracey

et al. 2001; Buckley et al. 2006; Gracey 2008). Briefly,

RNA from gill and muscle tissue was reverse transcribed to

cDNA and the resulting cDNAs were directionally cloned

into pTriplEx2 vector (Clontech; Mountain View, CA,

USA). Plasmid libraries were transformed into E. coli and

bacterial colonies were picked at random into 384-well

microtiter plates and grown overnight at 37�C. cDNA

inserts were amplified by PCR using 1 ll of bacterial

774 J Comp Physiol B (2011) 181:773–780

123

suspension in 50 ll standard PCR reactions with vector

specific primers. The PCR products generated from the

cDNA inserts isolated from the clones were sequenced

directly and sequencing was done from the 50 end using

primers specific to the plasmid vector. This process

generated thousands of clones that were identified via

BLAST-x searching against the NCBI public databases.

One of the generated sequences was for the gene of current

interest, c/ebp-d. This fragment (GenBank accession

number EB651785) includes much of the coding sequence

as well as a 200-bp region upstream from the start codon.

This region was screened for the existence of a consensus

STAT-3 binding site (TTACNGNGAA) and that of a

HIF-1a consensus binding site (ACGTG).

Quantification of protein concentration: solid-phase

immunochemistry

Concentrations of C/EBP-d protein in sampled tissues were

quantified via western blotting. Frozen tissue sections of

approximately 100 lg were thawed in 100 ll of homoge-

nization buffer, containing 32 mmol l-1 Tris–HCl (pH 6.8)

and 2% sodium dodecyl sulfate (SDS). Homogenates were

heated at 100�C for 5 min and centrifuged at 12,0009g for

10 min. Pellets were discarded, and total protein content of

the supernatants was determined by Bradford assay (Pierce,

Rockford, IL, USA). Ten micrograms of total protein from

each sample were separated on 10% gels by SDS-poly-

acrylamide gel electrophoresis. After separation, proteins

were transferred to nitrocellulose membranes by electro-

blotting at 30 V overnight at 4�C. Following transfer,

membranes were dried at 70�C for 45 min. Blots were

blocked for 1 h at room temperature (RT) in 5% non-fat

dry milk (NFDM) in 19 phosphate buffered saline (PBS)

under constant shaking. Blots were washed 3 times for

5 min in 19 PBS containing 0.01% Tween-20. Following

washes, blots were incubated in primary antibody (1:1,000

dilution in 19 PBS containing 5% NFDM) for 1.5 h at RT.

For quantification of C/EBP-d protein concentration, a

custom antibody was manufactured in rabbit using a pep-

tide corresponding to a deduced 15-aa sequence from the N

terminus region of the G. mirabilis C/EBP-d protein as an

antigen (Affinity BioReagents, Rockford, IL, USA). Fol-

lowing incubation in primary antibody, blots were washed

3 9 10 min in 19 PBS with 0.1% Tween-20, then incu-

bated for 1 h at RT in a secondary antibody (horseradish

peroxidase conjugated Protein G, BioRad, Hercules, CA,

USA) at a dilution of 1:5,000 in 19 PBS containing 5%

NFDM. Blots were washed 3 9 5 min in 19 PBS with

0.01% Tween-20, and exposed to enhanced chemilumi-

nescent reagent (ECL) (Amersham Pharmacia Biotech,

Piscataway, NJ, USA), for 5 min. Blots were wrapped in

plastic wrap and exposed to X-ray film (XOMAT-AR film,

Kodak, Rochester, NY, USA). Densitometry was con-

ducted on blots using ImageJ freeware (NCBI). The spec-

ificity of the antibody was determined as described in

Supplementary Materials (Fig. S1).

Statistics

The effect of ‘‘timepoint’’ on protein concentration was

determined by one-way ANOVA using GraphPad Prism

software (GraphPad Software, Inc., La Jolla, CA, USA).

Differences between values at a given timepoint and those

at time 0 were determined via post-hoc Dunnet’s multiple

comparison test. In the thermal acclimation experiment,

differences in the mean protein concentration between the

15�C and the 25�C group were determined via Student’s

t test.

Results

Sequencing of a 641-bp fragment of the G. mirabilis

C/EBP-d cDNA and a 200-bp portion of the 50 UTR of the

Gillichthys mirabilis mirabilis c/ebp-d revealed a STAT-3

binding site, also termed the ‘‘acute phase response

element’’ (APRE), which is the consensus sequence

50-TTACNGNGAA-30, located at the -120-bp position

(Fig. 1). Three hypoxia response element (HRE) motifs

(50-ACGTG-30) were also mapped to the 50UTR (Fig. 1).

SDS-polyacrylamide gel electrophoresis revealed that the

molecular weight of the G. mirabilis C/EBP-d protein is

approximately 35 kDa (Fig. 2).This molecular weight is

similar to that reported for other vertebrates (e.g. in rat,

Reinhold and Ekstrom 2004).

Levels of C/EBP-d protein increased in response to heat

exposure in all three tissues examined (Fig. 3a–c; ANOVA

p \ 0.0001 in all tissues). In the gill, there were two peaks

in C/EBP-d production, the first occurring at the

240–270 min timepoints and the second after 480 min

(Fig. 3a). In the white muscle, C/EBP-d concentration rose

after 180 min of heat shock and remained elevated above

control levels for the duration of the heat exposure, peaking

at 480 min (Fig. 3b). The response in the liver was more

delayed, with levels of C/EBP-d increasing after 240 min

Fig. 1 The 200-bp sequence upstream of the start codon of the

Gillichthys mirabilis c/ebp-d gene. The acute phase response element

(APRE) is located at the –120 bp position (in bold). Three hypoxia

responsive elements (HREs) are underlined

J Comp Physiol B (2011) 181:773–780 775

123

and then remaining elevated over controls for the duration

of the experiment (Fig. 3c). The magnitude of the increases

in C/EBP-d differed among tissue types. In the gill, levels

increased by approximately 100-fold over control values,

while in the white muscle and liver, the increases were on

the order of 6- and 1.75-fold, respectively.

In the thermal acclimation experiment, C/EBP-d levels

were significantly higher in the warm-acclimated (25�C)

group than in the cold-acclimated (15�C) group in all three

tissues (Fig. 4). In gill and white muscle, the mean con-

centration of C/EBP-d was more than 29 greater than that

in warm-acclimated individuals (t test p \ 0.0001). In the

liver, the difference was more moderate, as the mean level

of C/EBP-d in the warm-acclimated group was 1.29 as

great as that in the cold-acclimated group (t test p \ 0.05).

Discussion

The current study investigated the effect of both sub-lethal

heat stress and warm-acclimation on the production of a

known regulator of cell differentiation and cell cycle pro-

gression, C/EBP-d, in the gill, white muscle and liver of the

eurythermal goby, G. mirabilis. The salient findings were:

(1) heat stress caused significant increases in the

concentration of C/EBP-d in all three tissues, (2) the

kinetics and magnitude of its production were highly tis-

sue-specific, occurring on the order of minutes to hours, (3)

C/EBP-d production responded to a 1-month thermal

acclimation treatment in all three tissues, with higher levels

being measured in the warm-acclimated group. Further-

more, the presence of a putative STAT-3 binding site, the

APRE, and three putative HIF-1 binding sites, were iden-

tified in the 50-untranslated region (UTR) of the G. mira-

bilis c/ebp-d gene. It is possible, based on the known roles

that C/EBP-d plays in other vertebrates, that the up-regu-

lation of this gene observed here may be linked to stress-

related cessation of cell proliferation and/or the initiation of

apoptosis. If so, this would represent a novel pathway

governing the coordinated cellular response to heat stress a

marine poikilotherm.

4802400

a

b

c

Time at 32˚C (min)

Fig. 2 Representative western blots depicting C/EBP-d protein from

the (a) gill, (b) white muscle and (c) liver tissues of Gillichthysmirabilis. Lanes 1, 2 and 3 are samples exposed to 0, 240 or 480 min

of heat shock at 32�C, respectively. Fifteen micrograms of total

protein from each sample were separated on a 10% SDS-poly-

acrylamide gel and electrotransferred to nitrocellulose membranes.

Blots were probed with an a-C/EBP-d primary antibody, followed by

an HRP-conjugated secondary antibody and visualized via enhanced

chemiluminescence. Black arrows represent the location of a 30-kDa

molecular weight marker

0

20

40

60

80

100

120

140

0

2

4

6

8

10

0

0.5

1

1.5

2

2.5

Gill

White Muscle

Liver

*

* * *

**

** **

** ** **

*

*

*

a

b

c

4803602702401800Time at 32°C (min)

C/E

BP-

con

cent

ratio

n (R

elat

ive

OD

Uni

ts)

Fig. 3 The effect of exposure to heat stress (32�C) on levels of

C/EBP-d in (a) gill, (b) white muscle and (c) liver tissue from

Gillichthys mirabilis. Concentrations of C/EBP-d in n = 5 individuals

from each timepoint were measured via western blotting with a

custom primary a-C/EBP-d antibody. The effect of heat treatment was

significant in all three tissues (ANOVA; p \ 0.0001). Values at each

timepoint were normalized to the average of the time 0 values. Mean

and SD are reported. *Significant difference of means at a given

timepoint versus time 0 at p value \ 0.01. **Significant difference of

means at p value \ 0.001

776 J Comp Physiol B (2011) 181:773–780

123

Characterization of the G. mirabilis c/ebp-d cDNA

The C termini of the C/EBP family are highly conserved

among isoforms, consisting of a domain of basic amino

acids that mediates DNA-binding activity (Johnson 2005).

The C terminus also contains a leucine zipper, which is

responsible for dimer formation (Landschulz et al. 1988).

Formation of homodimers is a necessary step that precedes

acquisition of transactivation potential (Vinson et al. 1989)

and the dimerization domain is accordingly well-conserved

among C/EBP isoforms. There is, however, considerable

sequence divergence among isoforms at the N terminus. As

this region of the molecule contains its transactivation and

regulatory domains, it is likely here that the observed

specificity in the activity, regulation and function of vari-

ous C/EBP isoforms is maintained.

One established regulatory pathway governing de novo

C/EBP-d mRNA expression in mammals involves STAT-3

(Clarkson et al. 2006). It has been shown, for instance, that

the induction of C/EBP-d in mammary epithelial cells

treated with the inflammatory cytokine interleukin 6 (IL-6)

is dependent upon a signaling pathway that includes the

activation, through phosphorylation, of STAT-3 (Cantwell

et al. 1998). The existence of a STAT-3 binding site (the

APRE) in the 50 UTR of the G. mirabilis c/ebp-d gene

(Fig. 1) supports a conserved role for STAT-3 as a regu-

lator of vertebrate C/EBP-d. An important caveat to these

findings is that the role of regulatory elements located in

the 50-UTR of a given gene need to be directly established

as their proximity to the start codon can in some cases

render them non-functional.

Three binding sites for hypoxia inducible factor (HIF-1)

were also located in the G. mirabilis c/ebp-d 50 UTR

(Fig. 1). It should be noted that a HIF-1 ancillary sequence

(HAS)—a necessary downstream sequence for proper HIF-

1 binding in some vertebrate genes (Kimura et al. 2001)—

was not found in the 50UTR of G. mirabilis. HIF-1 is the

major regulator of the cellular response to reduced oxygen

tension (Wenger 2002), but has also been shown to be

responsive to both heat and cold (e.g. Katschinski et al.

2002; Rissanen et al. 2006). The deleterious effects that

both hypoxia and thermal stress can have on physiological

systems have long been acknowledged (reviewed in Wood

1991) and it is possible that HIF-1 plays an important role

in coordinating the cellular stress response to both stress-

ors. Additionally, it has been shown in mammalian cells

that cues such as exposure to growth factors, cytokines and

hormones that are known to induce C/EBP isoforms can

also induce HIF-1 (Richard et al. 2000; Page et al. 2002;

Ma et al. 2004). The ability of cell cycle regulatory genes

to act as both temperature and oxygen sensors in fishes

would be consistent with the roles that temperature and

oxygen availability play as the two most critical abiotic

variables governing metabolism and growth rates in these

species. Although it remains to be shown that hypoxia per

se is sufficient to induce C/EBP-d in fishes, HIF-1-medi-

ated signaling appears to be a potential pathway conferring

oxygen sensitivity to the G. mirabilis c/ebp-d gene.

In addition to the APRE, binding sites for other tran-

scription factors including NF-kB, cAMP responsive ele-

ment binding protein (CREB) and Sp1 have also been

found in promoters of mammalian C/EBP-d genes (Zhang

et al. 2007). While these binding sites were not found in

the 200-bp region proximal to the start codon from the

G. mirabilis c/ebp-d gene, they may exist further upstream.

The effect of heat stress on levels of C/EBP-d

In each of the tissues examined, exposure to heat resulted

in a significant increase in the concentration of C/EBP-dprotein (Fig. 3a–c). The timing and magnitude of the

increases varied considerably among the tissues, however.

In the gill tissue (Fig. 3a), levels of C/EBP-d rose imme-

diately and showed two peaks, one occurring at the

240–270 min timepoints and a second at 480 min. In

contrast to the gill, in both white muscle and liver, the

production of C/EBP-d protein increased steadily during

the heat shock exposure, reaching a maximum level at

480 min (Fig. 3b–c). These patterns raise the possibility

that the gill tissue, exposed directly to the external thermal

environment, may respond more dynamically to heat stress

exposure than other tissues such as the muscle and liver.

Few studies have directly tested the effect of hyper-

thermia on C/EBP-d expression in vertebrate cells, as most

attention has been paid to its expression and activity in

mammalian cells where environmental temperature is often

0

0.5

1

1.5

2

2.5

315 C

25 C

Gill White Muscle Liver

C/E

BP-

δco

ncen

trat

ion

(Rel

ativ

e O

D U

nits

) ** **

*

˚˚

Fig. 4 The effect of thermal acclimation to either 15�C (gray bars) or

25�C (white bars) for 4 weeks, on the concentration of C/EBP-dprotein in gill, white muscle and liver tissue from Gillichthysmirabilis. Concentrations of C/EBP-d in n = 5 individuals from each

timepoint were measured via western blotting. For a given tissue,

values were normalized to the mean of the 15�C group. Mean ± SD

is reported. *Significant difference between means of 15 versus 25�C

acclimation groups at a p value \ 0.05. **Significant difference in

means at p value \ 0.0001

J Comp Physiol B (2011) 181:773–780 777

123

considered constant. However, in a study on the effect of

elevated temperature on gene expression in mouse liver,

levels of c/ebp-d mRNA were unaffected by heat (Yiangou

et al. 1998). Despite not being responsive to heat stress in

human intestinal cells, c/ebp-d was inducible by exposure

to the proteasome inhibitor, MG-132 (Hungness et al.

2002). MG-132 is a potent chemical inducer of the heat

shock response (HSR) (Kim et al. 1999), as blocking the

proteasome results in the intracellular accumulation of

damaged protein, a condition that also results from expo-

sure to sufficiently elevated temperature. The induction of

c/ebp-d by proteasome inhibition, therefore, suggests that if

this cell cycle regulator does not respond directly to heat

stress in mammals, it can respond to one of its effects: the

appearance in the cell of abnormally folded proteins. It is

possible that a similar mechanism is at work here, as the

presence of denatured protein in the cell is sufficient to

induce other cellular stress responses, such as the HSR,

even in the absence of heat (Hightower 1980).

Evidence of the effect of heat stress on c/ebp-dexpression in other fish species is scarce. Interestingly, in a

tropical species of damselfish, Pomacentrus moluccensis,

transfer from 26 to 34�C resulted in a 1.9-fold repression of

c/ebp-d rather than an induction (Kassahn et al. 2007). The

b isoform was heat-inducible in the gill of heat-shocked

individuals of the cold-adapted Antarctic rock cod,

Trematomus bernacchii (Buckley and Somero 2009).

Identifying the mechanisms responsible for the disparate

patterns of response in the Antarctic and tropical species

and those reported for the estuarine species in the current

study requires further investigation.

In another transcriptional profiling study employing the

G. mirabilis microarray, c/ebp-d was found to be inducible

in the gills of individuals exposed to hyperosmotic stress

(Evans and Somero 2008). These findings, together with

the identification of HIF-1 binding sites described above,

are consistent with the hypothesized role of c/ebp-d as a

generally stress-responsive gene that may respond not only

to heat stress but also to other abiotic insults such as

osmotic stress and hypoxia.

The current study focuses on the d isoform of C/EBP,

but it should be noted that there is evidence for thermal

regulation of other isoforms in aquatic ectotherms. In the

symbiotic anemone Anthopleura elegantissima, c/ebp-bwas induced by heat stress but down-regulated in response

to UV exposure (Richier et al. 2008). In the same study, the

gene encoding another isoform, C/EBP-a, was down-

regulated during heat exposure. In another symbiotic

cnidarian, the coral Montastraea faveolata, thermal stress

repressed the expression of c/ebp-b (Desalvo et al. 2008).

The regulatory pathways controlling the expression and the

functions of the various C/EBP isoforms in stressed aquatic

species need further study.

The effect of thermal acclimation on the production

of C/EBP-d

C/EBP-d levels also varied between two thermal acclima-

tion groups, with significantly greater levels being mea-

sured in the tissues of individuals acclimated for a month to

25�C compared to those held at 15�C (Fig. 4). In all three

tissues, levels of C/EBP-d were higher in the warm-accli-

mated group. A similar finding has also been reported in

the bluefin tuna, Thunnus orientalis, with the expression of

c/ebp-d mRNA being greater in warm-acclimated individ-

uals than in the cold-acclimated group (Castilho et al.

2009). It is possible that the greater levels of C/EBP-d in

warm-acclimated fishes may be the result of repeated heat-

induction events, resulting in higher standing stocks of this

protein in the cell. Similar patterns are seen in another

thermally responsive pathway, the well-studied HSR, in

which individuals with a warmer recent thermal history

display higher standing stocks of heat shock proteins than

their colder acclimated counterparts (reviewed in Feder and

Hofmann 1999).

Evidence for a role of C/EBP-d in inducing cell cycle

arrest

While the role that C/EBP-d plays during heat stress remains

to be established, several lines of evidence support the

capacity of C/EBP-d to effect the arrest of the cell cycle in

response to external cues in other vertebrates (Johnson

2005). It has been demonstrated in mammary epithelial cells,

for example, that the over-expression of C/EBP-d results in

the onset of cell cycle arrest (O’Rourke et al. 1999). Fur-

thermore, inhibition of C/EBP-d by anti-sense c/ebp-dmRNA prevented the cell cycle arrest that is the normal

result of the removal of EGF or the application of IL-6. In our

previous study, the peaks in expression of c/ebp-d mRNA in

G. mirabilis gill tissue were inversely correlated with the

expression of three markers of progression through the G1/S

phase transition of the cell cycle, the histones H2B and H4

(Fig. S2; adapted from Buckley et al. 2006).

A hypothesized role for C/EBP-d in the cellular

response to heat stress in fishes

The ability to induce cell cycle arrest in response to heat

stress may be adaptive in organisms such as fishes, which

are poikilotherms that inhabit thermally variable environ-

ments, representing a method of shunting energy away

from cell growth and proliferation and toward the rescue

and repair mechanisms characteristic of the chaperone-

mediated HSR. This idea is consistent with our emerging

778 J Comp Physiol B (2011) 181:773–780

123

understanding of the existence of a highly conserved cellular

response to environmental stress in such organisms (Kultz

2005). Regardless of taxa, at the cellular level, the reaction

to environmental insult from various stressors can be defined

by a set of stereotyped responses, which, in concert, com-

prise the cellular stress response (CSR) (Kultz 2005). The

hallmarks of this response are (1) the immediate protection

of the cellular macromolecules such as proteins, through the

activity of molecular chaperones, (2) an increase in meta-

bolic output and a redistribution of energetic resources away

from routine housekeeping functions and toward stress

responses, (3) the temporary and reversible arrest of the cell

cycle, and (4) in extreme cases, apoptosis.

Clearly, coordinating such a broad suite of cellular

processes will rely on an equally broad spectrum of mol-

ecules and pathways. The findings reported here support

the inclusion of C/EBP-d as a potential member of the

group of genetic ‘‘master switches’’ that control the CSR,

with the hypothesized role of C/EBP-d being to arrest the

cell cycle during stress, thereby allowing the critical cyto-

protective functions of the CSR to proceed before normal

cell cycle progression is resumed. An alternative or perhaps

complimentary role may be to promote apoptosis in

response to stress exposures sufficient to cause DNA

damage or other macromolecular degradation. A greater

understanding of the regulation of C/EBP-d by heat and

other stressors, through its upstream mediators, may pro-

vide a mechanism by which environmental temperature

stress is linked to changes in energy resource allocation, a

physiological consideration which can in turn influence

critical ecological processes.

Acknowledgments This research was supported by the Oregon

Health Sciences University Medical Research Foundation grant

#0801 to BAB. The author thanks Dr. George Somero for use of

laboratory space at Stanford University’s Hopkins Marine Station for

the heat exposures.

References

Buckley BA, Hofmann GE (2002) Thermal acclimation changes

DNA-binding activity of heat shock factor 1 (HSF1) in the goby

Gillichthys mirabilis: implications for plasticity in the heat-

shock response in natural populations. J Exp Biol

205:3231–3240

Buckley BA, Somero GN (2009) cDNA microarray analysis reveals

the capacity of the cold-adapted Antarctic fish Trematomusbernacchii to alter gene expression in response to heat stress.

Polar Biol 32:403–415

Buckley BA, Gracey AY, Somero GN (2006) The cellular response to

heat stress in the goby Gillichthys mirabilis: a cDNA microarray

and protein-level analysis. J Exp Biol 209:2660–2677

Cantwell CA, Sterneck E, Johnson PF (1998) Interleukin-6-specific

activation of the C/EBP-d gene in hepatocytes is mediated by

Stat3 and Sp1. Mol Cell Biol 18:2108–2117

Castilho P, Buckley BA, Somero GN, Block BA (2009) Heterologous

hybridization to a cDNA microarray reveals the effect of thermal

acclimation in the endothermic bluefin tuna, (Thunnus oriental-is). Mol Ecol 18:2092–2102

Clarkson RWE, Boland MP, Kritikou EA, Lee JM, Freeman TC,

Tiffen PG, Watson CJ (2006) The genes induced by signal

transducer and activators of transcription (STAT)3 and STAT5

in mammary epithelial cells define the roles of these STATs in

mammary development. Mol Endocrinol 20(3):675–685

Dearth LR, DeWille J (2003) Post-transcriptional and post-transla-

tional regulation of C/EBPd in G0 growth-arrested mammary

epithelial cells. J Biol Chem 278(13):11246–11255

Desalvo MK, Voolstra CR, Sunagawa S, Schwarz JA, Stillman JH,

Coffroth MA, Szmant AM, Medina M (2008) Differential gene

expression during thermal stress and bleaching in the Caribbean

coral Montastrea faveolata. Mol Ecol 17:3952–3971

Evans T, Somero GN (2008) A microarray-based transcriptomic time-

course of hyper- and hypoosmotic signaling events in the

euryhaline fish Gillichthys mirabilis: osmosenors to effectors.

J Exp Biol 211:3636–3649

Feder ME, Hofmann GE (1999) Heat-shock proteins, molecular

chaperones, and the stress response: evolutionary and ecological

physiology. Annu Rev Physiol 61:243–282

Gery S, Koeffler HP (2009) Per2 is a C/EBP target gene implicated in

myeloid leukemia. Integr Cancer Ther. doi:10.1177/1534735

409352084

Gery S, Gombart AF, Yi WS, Koeffler C, Hofmann W-K, Koeffler HP

(2005) Transcriptional profiling of C/EBP targets identifies Per2

as a gene implicated in myeloid leukemia. Neoplasia

106:2827–2836

Gracey AY (2008) The Gillichthys mirabilis Cooper array: a platform

to investigate the molecular basis of phenotypic plasticity. J Fish

Biol 72:2118–2132

Gracey AY, Troll JV, Somero GN (2001) Hypoxia-induced gene

expression profiling in the euryoxic fish Gillichthys mirabilis.

Proc Nat Acad Sci 98:1993–1998

Hightower LE (1980) Cultured animal cells exposed to amino acid

analogues or puromycin rapidly synthesize several polypeptides.

J Cell Physiol 102(3):407–427

Hungness ES, Robb BW, Luo G-j, Hershko DD, Hasselgren P-O

(2002) Hyperthermia-induced heat shock activates the transcrip-

tion factor C/EBP-b and augments IL-6 production in human

intestinal epithelial cells. J Am Coll Surg 195(5):619–626

Johnson PF (1993) Identification of C/EBP basic region residues

involved in DNA sequence recognition and half-site space

preference. Mol Cell Biol 13(11):6919–6930

Johnson PF (2005) Molecular stop signs: regulation of cell-cycle

arrest by C/EBP transcription factors. J Cell Sci 118:2545–2555

Kassahn KS, Caley MJ, Ward AC, Connolly AR, Stone G, Crozier

RH (2007) Heterologous hybridization to study the early gene

response to heat stress in a coral reef fish. Mol Ecol 16:1749–

1763

Katschinski DM, Le L, Heinrich D, Wagner KF, Hofer T, Schindler

SG, Wenger RH (2002) Heat induction of the unphosphorylated

form of hypoxia inducible factor-1a is dependent on heat shock

protein-90 activity. J Biol Chem 277(11):9262–9267

Kim D, Kim S-H, Li GC (1999) Proteasome inhibitors MG132 and

lactacystin hyperphosphorylate HSF1 and induce hsp70 and

hsp27 expression. Biochem Biophys Res Comm 254:264–268

Kimura H, Weisz A, Ogura T, Hitomi Y, Kurashima Y, Hashimoto K,

D’Acquisto F, Makuuchi M, Esumi H (2001) Identification of

hypoxia-inducible factor 1 ancillary sequence and its function in

vascular endothelial growth factor gene induction by hypoxia

and nitric oxide. J Biol Chem 276(3):2292–2298

Kultz D (2005) Molecular and evolutionary basis of the cellular stress

response. Ann Rev Physiol 67:225–257

J Comp Physiol B (2011) 181:773–780 779

123

Landschulz WH, Johnson PF, McKnight SL (1988) The leucine

zipper: a hypothetical structure common to a new class of DNA

binding proteins. Science 240:1759–1764

Lekstrom-Himes J, Xanthopolous KG (1998) Biological role of the

CCAAT/enhancer-binding protein family of transcription fac-

tors. J Biol Chem 273:28545–28548

Lyons SE, Shue BC, Lei L, Oates AC, Zon LI, Liu PP (2001)

Molecular cloning, genetic mapping, and expression analysis of

four zebrafish c/ebp genes. Gene 281:43–51

Ma Y, Freitag P, Zhou J, Brune B, Frede S, Fandrey J (2004) Thyroid

hormone induces erythropoietin gene expression through aug-

mented accumulation of hypoxia-inducible factor-1. Am J Phys

287:R600–R607

Milde-Langosch K, Loning T, Bamberger A-M (2003) Expression of

the CCAAT/enhancer-binding proteins C/EBPa, C/EBPb and

C/EBPd in breast cancer: correlations with clinicopathologic

parameters and cell-cycle regulatory proteins. Breast Cancer Res

Treat 79:175–185

Miller M, Shuman JD, Sebastian T, Dauter Z, Johnson PF (2003)

Structural basis for DNA recognition by the basic region leucine

zipper transcription factor CCAAT/Enhancer-binding protein

alpha. J Biol Chem 278(17):15178–25284

Nerlov C (2007) The C/EBP family of transcription factors: a

paradigm for interaction between gene expression and prolifer-

ation control. Trends Cell Biol 17(7):318–324

O’Rourke JP, Newbound GC, Hutt JA, DeWille J (1999) CCAAT/

enhancer-binding protein d regulates mammary epithelial cell Go

growth arrest and apoptosis. J Biol Chem 23:16582–16589

Osada S, Yamamoto H, Nishihara T, Imagawa M (1996) DNA

binding specificity of the CCAAT/Enhancer-binding protein

transcription factor family. J Biol Chem 271(7):3891–3896

Page EL, Robitaille GA, Pouysseger J, Richard DE (2002) Induction

of hypoxia-inducible factor-1 alpha by transcriptional and

translational mechanisms. J Biol Chem 277:48403–48409

Porter DA, Krop IE, Nasser S, Sgroi D, Kaelin CM, Marks JR,

Riggins G, Polyak K (2001) A SAGE (serial analysis of gene

expression) view of breast tumor progression. Cancer Res

61:5697–5702

Porter DA, Lahit-Domenici J, Keshaviah A, Bae YK, Argani P, Marks

J, Richardson A, Cooper A, Strausberg R, Riggins GJ, Schnitt S,

Gabrielson E, Gelman R, Polyak K (2003) Molecular markers in

ductal carcinoma in situ of the breast. Mol Cancer Res

1:362–375

Ramji DP, Foka P (2002) CCAAT/enhancer-binding proteins:

structure, function and regulation. Biochem J 365:561–575

Reinhold AC, Ekstrom J (2004) Expressions of C/EBP-enhancer-

binding proteins and c-Myc in the parotid gland of the rat: in

vivo effects of isoprenaline, bethanechol, vasoactive intestinal

peptide and food intake. Arch Oral Biol 49(5):345–354

Richard DE, Berra E, Pouyssegur J (2000) Nonhypoxic pathway

mediates the induction of hypoxia-inducible factor 1a in vascular

smooth muscle cells. J Biol Chem 275:26765–26771

Richier S, Rodriguez-Lanetty, Schnitzler CE, Weis VM (2008)

Response of the symbiotic cnidarian Anthopleura elegantissimatranscriptome to temperature and UV increase. Comp Biochem

Physiol D 3:283–289

Rissanen E, Tranberg HK, Sollid J, Nilsson GE, Nikinmaa M (2006)

Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a

poikilothermic vertebrate, crucian carp (Carassius carassius).

J Exp Biol 209:994–1003

Rosenbauer F, Tenen DG (2007) Transcription factors in myeloid

development: balancing differentiation with transformation.

Nature Rev Immunol 7(2):105–117

Sivko GS, DeWille JW (2004) CCAAT/enhancer binding protein d(C/EBP- d) regulation and expression in human mammary

epithelial cells: I. ‘‘Loss of function’’ alterations in the C/EBP- dgrowth inhibitory pathway in breast cancer cell lines. J Cell

Biochem 93:830–843

Sivko GS, Sanford DC, Dearth LD, Tang D, DeWille JW (2004)

CCAAT/enhancer binding protein d (C/EBP- d) regulation and

expression in human mammary epithelial cells: II. Analysis of

activating signal transduction pathways, transcriptional, post-

transcriptional, and post-translational control. J Cell Biochem

93:844–856

Tang D, Sivko GS, DeWille J (2006) Promoter methylation reduces

C/EBPd (CEBPD) gene expression in the SUM-52PE human

breast cancer cell line and in primary breast tumors. Breast

Cancer Res Treat 95:161–170

Thangaraju M, Rudelius M, Bierie B, Raffeld M, Sharan S,

Hennighhausen L, Huang A-M, Sterneck E (2005) C/EBP-d is

a crucial regulator of pro-apoptotic gene expression during

mammary gland involution. Development 132(21):4675–4683

Vinson CR, Sigler PB, McKnight SL (1989) Scissors-grip model for

DNA recognition by a family of leucine zipper proteins. Science

246:911–916

Wedel A, Zeigler-Heitbrock HW (1995) The C/EBP family of

transcription factors. Immunobiology 193:171–185

Wenger RH (2002) Cellular adaptation to hypoxia: O2-sensing protein

hydroxylases, hypoxia-inducible transcription factors, and O2-

regulated gene expression. FASEB J 16:1151–1162

Wood SC (1991) Interactions between hypoxia and hypothermia. Ann

Rev Physiol 53:71–85

Yiangou M, Paraskeva E, Hsieh C-C, Markou E, Victoratos P,

Scouras Z, Papaconstantinou J (1998) Induction of a subgroup of

acute phase protein genes in mouse liver by hyperthermia.

Biochim Biophys Acta 1396:191–206

Zhang Y, Sif S, DeWille J (2007) The mouse C/EBPdelta gene

promoter is regulated by STAT3 and Sp1 transcriptional

activators, chromatin remodeling and c-Myc repression. J Cell

Biochem 102(5):1256–1270

Zhang Y, Liu T, Yan P, Huang T, DeWille J (2008) Identification and

characterization of CCAAT/Enhancer Binding Protein delta (C/

EBPdelta) target genes in G0 growth arrested mammary

epithelial cells. BMC Mol Biol 9:83

780 J Comp Physiol B (2011) 181:773–780

123