www.elsevier.com/locate/devbrainres

Developmental Brain Resea

Research Report

Butyrate, a gut-derived environmental signal, regulates tyrosine

hydroxylase gene expression via a novel promoter element

Pranav Patel, Bistra B. Nankova, Edmund F. LaGamma*

Division of Newborn Medicine, Department of Pediatrics, New York Medical College, Valhalla, NY 10595, USA

The Regional Neonatal Center, The Maria Fareri Children’s Hospital, Westchester Medical Center, Valhalla, NY 10595, USA

Accepted 12 August 2005

Available online 13 September 2005

Abstract

Butyrate is a diet-derived, gut fermentation product with an array of effects on cultured mammalian cells including inhibition of

proliferation, induction of differentiation and regulation of gene expression. We showed that physiological concentrations of butyrate can

regulate transcription of tyrosine hydroxylase (TH) and preproenkephalin (ppEnk) gene in PC12 cells. In promoter deletion studies,

electrophoretic mobility shift assays and by site-directed mutagenesis, we identified a novel butyrate response element (BRE) in the 5Vupstream region of the rat TH gene, homologous to the previously mapped motif in the ppEnk promoter. No such enhancers were found in

DBH or PNMT promoters, and both catecholamine system-related gene promoters were unaffected by butyrate. The BRE motif interacts with

nuclear proteins in a sequence-specific manner, shows binding potentiation in butyrate-differentiated PC12 cells and bound protein(s) are

competed away with TH-CRE oligonucleotides or by the addition of CREB-specific antibodies, suggesting involvement of CREB or CREB-

related transcription factors. Moreover, single point mutation in the distal BRE abolished binding of transcription factors and reduced the

response to butyrate in transient transfection studies. The canonical CRE motif of the TH promoter was also found necessary for

transcriptional activation of the TH gene by butyrate. Our data identified a novel functional element in the promoter of both the TH and

ppEnk genes mediating transcriptional responses to butyrate. Dietary butyrate may have an extended role in the control of catecholamine and

endogenous opioid production at the level of TH and ppEnk gene transcription neuronal plasticity, cardiovascular functions, stress adaptation

and behavior.

D 2005 Elsevier B.V. All rights reserved.

Theme: Neurotransmitter, modulators, transporters and receptors

Topic: Catecholamines

Keywords: Tyrosine hydroxylase; Catecholamine; Short chain fatty acid; Butyrate response element; cAMP responsive element binding protein (CREB)

0165-3806/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.devbrainres.2005.08.005

Abbreviations: SCFA, short chain fatty acid; SB, sodium butyrate; BRE,

butyrate response element; PKA, protein kinase A; cAMP, cyclic AMP;

CREB, cAMP responsive element binding protein; CRE, cAMP responsive

element; IP3, inositol triphosphate; EMSA, electrophoretic mobility shift

assay; TH, tyrosine hydroxylase; PNMT, phenylethanolamine N-methyl

transferase; DBH, dopamine h-hydroxylase; ppEnk, preproenkephalin;

MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal

regulated kinase; PC12, pheochromocytoma cell line; CAT, chlorampheni-

col acetyl transferase

* Corresponding author. Department of Pediatrics, New York Medical

College, Valhalla, NY 10595, USA. Fax: +1 914 493 1488.

E-mail address: edmund_lagamma@nymc.edu (E.F. LaGamma).

1. Introduction

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in

biosynthesis of the catecholamine transmitters dopamine,

norepinephrine and epinephrine [30,48]. In the intact

animal, TH synthesis is regulated by transsynaptic activity

associated with cholinergic innervation of structures like the

adrenal medulla [13,21,48]. Among various possibilities,

these transsynaptic signals utilize cAMP second-messenger

systems that converge on the cAMP response element

(CRE) and its binding proteins (CREB family) to increase

TH gene transcription and thus TH mRNA accumulation

[3,30,32]. After birth, the capacity of this transsynaptic

rch 160 (2005) 53 – 62

P. Patel et al. / Developmental Brain Research 160 (2005) 53–6254

regulatory process accelerates abruptly between 7 and 10

days postnatal age, an effect only partially explained by

maturing innervation and hormonal changes [11,21,30,

48,53].

Short chain fatty acids (SCFAs) like butyrate arise from a

diet-derived fermentation process created by newly acquired

gut flora and accumulate in the blood during the immediate

postnatal period [8,10,14,15,24,50,51]. What is significant

is that SCFAs produce a wide array of effects on cultured

mammalian cells including inhibition of proliferation,

induction of differentiation and induction or repression of

gene expression where many of those effects also involve

cAMP-dependent mechanisms [2,37,42].

We have previously shown that both TH and the

neuropeptide transmitter gene, preproenkephalin (ppEnk),

have promoters that are up-regulated in butyrate-treated

PC12 cells using a reporter gene system (chloramphenicol

acetyl transferase; CAT) in vitro [42]. In comparison, there

was no increase in reporter gene activity in response to

butyrate when controlled by either the rat DBH or PNMT

(dopamine h-hydroxylase or phenylethanolamine N-methyl

transferase) promoters [42]. Since only the TH or ppEnk

promoter sequences were driving expression of CAT, our

prior data suggested that butyrate was acting at selected

‘‘target’’ genes rather than causing a generalized activation

of transcription. Moreover, our previous work indicated that

cAMP-dependent mechanisms were involved with butyrate-

induced accumulation of TH mRNA (DeCastro et al., Shah

et al., unpublished data, also in [37]).

In many butyrate-inducible genes, different DNA motifs

have been already associated with the butyrate response.

Those genes include the ppEnk (GC-rich motif 5VGCCTGGC. . . [23]), the CCAAT motif in the gamma-

globin [17,39], the Sp1-like enhancer in G alpha (i2) and the

tumor suppressor p21 [52,55]. Each of these systems also

shares cAMP regulatory pathways consistent with an

interaction between transcription factors.

In the present report, we sought to determine whether

butyrate acts by inducing binding of specific transcription

factors to the TH promoter, and, if so, which elements

primarily mediate the butyrate effect on TH gene

expression.

2. Methods

2.1. PC12 cell model and transfection

PC12 cells, originally described by Lloyd Greene [19],

were obtained from Simon Halegoua (Department of

Neurobiology and Behavior, SUNY Stony Brook) and were

grown in DMEM supplemented with 10% horse serum, 5%

fetal bovine serum with 50 Ag/ml streptomycin and 50 IU/

ml penicillin in a humidified 37 -C and 10% CO2

atmosphere as described earlier [42] PC12 cells were treated

for desired periods of time (see figure legends) with

indicated doses of sodium butyrate (SB; Sigma-Aldrich,

St. Louis, MO).

Butyrate responsive elements in the TH gene were

analyzed by transient transfection of PC12 cells with

plasmid vectors containing various lengths of the 5Vpromoter sequences of the rat TH gene controlling the

expression of either chloramphenicol acetyl-transferase

(CAT, [34]) or luciferase (LUC) reporter genes. PC12 cells

(in quadruplicate) were either left untreated or treated with

sodium butyrate in media for 1 day. After 24 h, plasmids

were transfected into PC12 cells by electroporation at 300 V

and 500 AF, with 20 Ag of DNA per 100 mm plate (106–107

cells). Cells treated with butyrate continue to receive media

supplemented with 6 mM butyrate after electroporation.

After the indicated treatment and time interval, the cells

were harvested, and protein concentrations in total cell

lysates were determined [6]. CAT reporter gene activity was

determined by the liquid scintillation method [47], and LUC

activity was measured by luminometry, as recommended by

the manufacturer (Promega, Madison, WI).

2.2. Site-directed mutagenesis

The 5V segment of the rat TH promoter (�773/+27 bp)

was inserted into a pGL3-Basic vector (Promega, Madison,

WI) expressing the firefly luciferase reporter gene. The

desired mutation of the putative BRE site (cytosine to

adenine; C-to-A alteration at position �507) was performed

using the ExSiteTM PCR-based site-directed mutagenesis kit

and instructions by Stratagene (La Jolla, CA). The same

approach was used to create a G-to-A single point mutation

at position �41 of the canonical CRE motif. The mutations

were confirmed by sequencing.

2.3. Preparation of nuclear extracts

Nuclear extracts from control and butyrate-treated PC12

cells were prepared according to the method of Dignam et al.

[12]. To all solutions, a cocktail of protein degradation and

phosphatase inhibitors were added (Sigma, St. Louis, MO) to

a final concentration of: 2 mM benzamidine, 5 Ag/ml

leupeptine, 5 Ag/ml pepstatine, 0.5 mM PMSF, 0.5 mM

DTT, 1 mM ammonium molybdate, 2 mM sodium fluoride

and 10 mM sodium pyrophosphate.

2.4. Electrophoretic mobility shift assay (EMSA)

DNA–protein binding reactions were carried out for 30

min at room temperature in 12% glycerol, 12 mM HEPES, 8

mM Tris–HCl, 1 mM EDTA, 1 mM DTT and 60 mM KCl.

The standard reaction includes 1 Ag BSA, 1 Ag poly(dI–dC),0.5 ng of 32P-end labeled ds oligonucleotide (30,000 cpm)

and nuclear extract (3 to 5 Ag protein) in a final volume of 15

Al as we previously described [40]. The sense and anti-sense

oligonucleotides spanning the CRE (TGACGTCA), putative

BRE (GCCTGGA) and putative BRE with single point

Fig. 1. (A) Schematic diagram of rat TH promoter showing multiple

previously described regulatory elements and the location of the TH-BRE.

(B) Plasmid constructs with sequential deletions in the TH promoter driving

the expression of a CAT reporter gene that were transfected into PC12 cells

in the presence or absence of 6 mM sodium butyrate (SB, 24 h treatment).

Cell extracts were prepared after another 24 h and assayed for CAT activity.

There is a progressive loss in reporter activity induction with sequential

deletions. Values represent means T SEM from quadruplicate cell cultures

and were compared with CAT activity in parallel cultures transfected with

identical plasmids treated with control media. Open bars = control; filled

bars = butyrate. *P < 0.05 from either basal levels (vehicle) or from �773

to �41 results.

P. Patel et al. / Developmental Brain Research 160 (2005) 53–62 55

mutation (GCATGGA) in the rat TH promoter were syn-

thesized (Invitrogen).

CRE: 5V . . . TGGGGGACCAGAGGGGCTTTGACGT-CAGCCT . . . 3VBRE: 5V . . . GTGTCTCATAGGACCTGCCTGGATC-CAGCCCCAG . . . 3VmBRE: 5 V . . . GTGTCTCATAGGACCTGCATG-

GATCCAGCCCCAG . . . 3V

The complementary oligonucleotides for each probe

were annealed, gel-purified and 32P-labeled using T4

DNA kinase (Invitrogen, Grand Island, NY) by following

manufacturer’s protocol. Approximately 30,000–50,000

cpm of radioactive probe (0.05–0.1 ng) and nuclear extracts

(3–5 Ag) in 12.5% glycerol, 12 mM HEPES, pH 7.9, 8 mM

Tris–HCl, pH 7.8, 1 mM EDTA, 1 mM DTT and 60 mM

KCl with 1 Ag of poly (dI–dC) in a final volume of 15 Alwere incubated for 30 min at room temperature in each

standard binding reaction as described previously [40].

Competition was performed by adding a 100-fold molar

excess of competitor non-radioactive oligonucleotides to the

reaction before the nuclear extracts or aCREB antibody

(sc240-X from Santa Cruz Biotechnology, Santa Cruz CA).

The DNA–protein complexes were resolved on 6% poly-

acrylamide gels in 0.25 � Tris–borate buffer. Subsequently,

the gels were fixed, vacuum-dried and autoradiographed

using intensifying screens and Kodak XAR-5 films [40].

2.5. Statistical analysis

Statistical significance was determined using Student’s t

test for experiments with two groups or by performing an

analysis of variance (ANOVA) followed by Fisher’s Least

Significant Difference Test for experiments with more than

two groups. A level of P < 0.05 was considered statistically

significant.

3. Results

3.1. Two regions in the rat TH promoter are important for

its transcriptional activation by butyrate

Endogenous TH and ppEnk mRNA accumulates in PC12

cells after treatment with butyrate [42], and in vitro

transfection with either a TH or ppEnk promoter-CAT

reporter construct yields similar results [42]. Together, these

data suggest that sufficient regulatory information exists in

either gene’s proximal promoter to confer butyrate respon-

siveness. Therefore, to determine specifically what regions

of the TH promoter were involved in mediating these

effects, 5V sequential deletion-reporter constructs were testedin the presence of 6 mM butyrate and assayed for reporter

gene activity relative to untreated cells transfected with the

same construct (Fig. 1).

When using the full-length TH promoter (�773/+27 bp),

butyrate treatment resulted in a 7-fold increase in CAT

activity (Fig. 1). Deletion of a 501 bp distal fragment (�773

through �272 bp) removing three AP2 binding sites [29]

was associated with a 30% reduction in responsiveness to

butyrate (Fig. 1). These data suggest that AP2 or other

previously unrecognized cis-acting regulatory element(s)

exist in this region of the TH distal promoter and are needed

for maximal activation of transcription by this SCFA.

A more proximal �272/+27 bp fragment of TH

promoter contains multiple other previously characterized

functional motifs including the hypoxia-inducible factor 1

(HIF1) site and AP1, AP2, E-box, octamer–heptamer, Sp1

and cAMP/calcium response elements [16,29,45,46,54]. To

determine whether any of these well-studied regulatory sites

were important modulators of TH promoter activity by

butyrate, we evaluated the effect of removal of an additional

164 bp of the TH promoter (from �272 to �108 bp). In

PC12 cells transfected with this p5VTH construct (�108/

+27), the observed increase in CAT activity following

butyrate treatment was similar to that obtained with the

longer p5VTH CAT plasmid (�272/+27), indicating that

there were no additional butyrate-dependent elements in this

region.

However, when the sequential deletion was extended

to �41 bp (which disrupted the canonical CRE site),

P. Patel et al. / Developmental Brain Research 160 (2005) 53–6256

there was no statistically significant difference in reporter

gene activity detected between control (vehicle) and

butyrate-stimulated PC12 cells. This suggested that a

region (or regions) of the TH promoter between �108

and �41 bp was required for the activation of tran-

scription by sodium butyrate. Interestingly, this segment

of the TH promoter contains the canonical cAMP

response element (CRE; 5VTGACGTCA, �45 to �38)

that is essential for its basal as well as cAMP/Ca2+-

inducible expression [22,28,32,41].

With this information, we conclude that there are at least

two separate upstream regions important for achieving

maximal TH promoter activation by butyrate in PC12 cells:

a distal segment from �773 through �272 bp and a second

proximal region (�108 to �41 bp) which contains the

canonical CRE enhancer.

3.2. Sequence homologies between TH and ppEnk promoter

elements mediating butyrate response

In the rat ppEnk promoter, La Gamma et al. identified

two regions (by a combination of sequential deletion

studies, foot print and gel shift analyses) essential for

transcriptional activation by butyrate: a distal GC-rich motif

at �452 bp (GCCTGGC; named BRE) and the two well-

characterized proximal ppEnk CREs between �102 and

�77 bp [31]. Based on the functional similarities in

upstream (¨500 bp) and CRE downstream regions, we

hypothesized that transcriptional activation by butyrate for

both TH and ppEnk genes may utilize a similar combination

of cis-acting factors involving a distal ‘‘BRE’’ site and a

proximal CRE enhancer.

Therefore, to determine whether a BRE element(s)

homologous to the sequence in the ppEnk promoter was

indeed present in the rat TH promoter, we performed a

‘‘BLAST’’ homology search for GCCTGGC (NCBI). We

found two matches, one residing in the �773 through �272

bp butyrate-responsive region of the TH promoter (Fig. 1)

with a second match at position �37 to �32 bp of the TH

promoter adjacent to the canonical CRE.

3.3. The distal BRE motif is involved in DNA–protein

interactions in vitro

To determine whether there were DNA–protein inter-

actions at the distal TH-BRE sequence, an oligonucleotide

spanning the TH-BRE element (position �509 to �504 bp)

of the rat TH promoter was radiolabeled and incubated with

nuclear extracts isolated from control (vehicle) and butyrate-

differentiated PC12 cells. The complexes were separated by

EMSA (Fig. 2).

With nuclear extracts from control, untreated PC12

cells, three DNA–protein complexes appeared (Fig. 2B,

lane 2). Incubation of nuclear extracts from butyrate-

treated cells with the radiolabeled oligonucleotides resulted

in formation of a new low mobility complex that was

nearly undetectable in control extracts and increased with

longer exposure to butyrate at 48 vs. 24 h (Fig. 2B,

compare lane 2 to lanes 3 and 4). All complexes were

competed away with the addition of a 100-fold excess of

non-labeled BRE oligonucleotides (lane 5, specific com-

petitor; ‘‘Sp’’), and none of the complexes was affected by

addition of the same quantity of scrambled oligonucleo-

tides (lane 7, nonspecific competitor; ‘‘Nsp’’).

3.4. CREB or CREB-related proteins bind to the distal BRE

in butyrate-differentiated cells

To determine whether the butyrate-induced protein–

DNA complex also had binding affinity at the TH-CRE,

we performed competition experiments with a TH-CRE

oligonucleotide (Fig. 2B, lane 6). The TH-CRE competitor

reduced the intensity of the higher molecular weight

complex, consistent with the involvement of CREB- or

ATF-like nuclear protein factor. To confirm that the

proteins bound to this upstream BRE oligonucleotide

sequence were indeed related to CREB or other CREB

family proteins, we added anti-CREB antibodies (aCREB)

to the reaction. aCREB resulted in a significant decrease

in intensity of the butyrate-induced upper band consistent

with binding of CREB or CREB-related proteins at this

position (Fig. 2C).

In order to confirm that the BRE motif of the TH

promoter is involved in sequence-specific DNA–protein

interactions, we generated a single point mutation in the

BRE sequence (GCCTGG Y GCATGG; mBRE). This

single nucleotide change within the BRE element com-

pletely abolished protein–DNA interactions in control

condition (Fig. 3, lanes 1 and 2) and greatly reduce them

in nuclear extracts isolated after exposure to butyrate (Fig. 3,

lanes 3 and 4), compared to results using the wild-type BRE

(Fig. 3, lane 5).

Together, our results identified a novel and previously

unrecognized motif in the distal TH promoter involved in

sequence-specific DNA–protein interactions with CREB or

CREB-related transcription factors that are potentiated in

butyrate-exposed PC12 cells.

3.5. Mutation of the BRE site reduces butyrate-induced TH

gene induction

In view of the mobility shift competition and antibody

data, we now sought to determine whether altering the

BRE element is functional in vivo. We used site-directed

mutagenesis to create the same single point mutation in the

BRE motif as in the EMSA experiments (GCCTGG YGCATGG) but using the full-length TH promoter (�773/

+27) driving expression of firefly luciferase. The mutated

BRE-TH-reporter construct or the wild-type TH promoter

plasmids were electroporated into PC12 cells. To half of

the cultures, butyrate was added as described in Methods,

and the reporter gene activity was compared (Fig. 4).

Fig. 2. (A) Sequence of the TH-BRE oligonucleotide probe used for EMSA. (B) Typical gel shift pattern with labeled BRE oligonucleotides and nuclear

extracts from control (C, lane 2) and butyrate (SB)-differentiated PC12 cells for 24 h (lane 3) or 48 h (lanes 4–7). Sp = specific competitor, 100� molar excess

of unlabeled BRE oligonucleotide; CRE = unlabeled oligonucleotide spanning TH-CRE site; Nsp = nonspecific competitor, 100� molar excess of scrambled

oligonucleotide. (C) EMSA radiograph utilizing a labeled TH-BRE oligonucleotide and nuclear extracts from butyrate-differentiated PC12 cells. When CREB-

specific antibody (aCREB) was included in the binding reaction (lane 3), the formation of the butyrate-induced complex was reduced consistent with

involvement of CREB or CREB-related proteins.

P. Patel et al. / Developmental Brain Research 160 (2005) 53–62 57

Mutation of the BRE motif resulted in a 50% reduction in

reporter gene activity in response to butyrate when compared

to the wild-type promoter (Fig. 4, P < 0.05) with no

significant change in basal levels of TH promoter activity.

This loss of function was consistent with a transcriptional

requirement for an intact BRE element to achieve butyrate

induction from the TH promoter.

To confirm that the mBRE reporter plasmid had in fact

remained functional, we treated PC12 cells with forskolin

(10 AM; an inducer of adenylate cyclase) in a similar

experiment paradigm (i.e. instead of butyrate). Forskolin

increased reporter gene activity to similar extent whether the

cells were transfected with either the wild-type promoter or

plasmids containing the mutated distal BRE motif (data not

Fig. 3. Representative EMSA with nuclear extracts from control (C) and PC12 cells treated with butyrate (SB) used to interact with the wild-type BRE

oligonucleotide (lane 5) or mutant BRE oligonucleotide (M1, lanes 1–4) probe. Single point mutation at the BRE site resulted in complete loss of the DNA–

protein interactions.

P. Patel et al. / Developmental Brain Research 160 (2005) 53–6258

shown). These results support the conclusion that the BRE

motif binds nuclear protein and is a functional promoter

element of the TH gene essential for maximal responses to

butyrate.

3.6. Mutation of the canonical CRE site in the TH promoter

attenuates the response to butyrate

Previous reports indicate that butyrate effects involve

cAMP-dependent intracellular mechanisms [2,37,42]. We

now sought to determine whether the canonical CRE

Fig. 4. A single nucleotide change was introduced either in the distal BRE site or t

the cartoon. Both, wild-type and mutant promoter constructs were transiently trans

as described in Methods. Bars represent the results of the TH promoter driving exp

(filled bars). *P < 0.05 from either basal levels or from BRE and CRE single po

respective basal level controls (vehicle).

element was directly involved in butyrate-mediated induc-

tion from the TH promoter. To accomplish this, a single

point mutation was created in the TH-CRE sequence

(TGACGTCA Y TGACATCA, position �41), and tran-

sient transfection experiments were performed. Both wild-

type and mutant promoter constructs were electroporated

into PC12 cells, and the response to butyrate was examined.

The mutant CRE resulted in a lower basal level of reporter

gene activity similar to results obtained when using protein

kinase A-deficient PC12 cells (see for example [35] and

references within). Moreover, the mutant CRE was also

he canonical CRE of the rat TH promoter construct (�773/+27) as shown in

fected into PC12, cells and the response to butyrate was examined after 24 h

ression of firefly luciferase in basal (open bars) or butyrate-exposed cultures

int mutations. Values represent mean T SEM and were normalized to their

P. Patel et al. / Developmental Brain Research 160 (2005) 53–62 59

associated with a >80% reduction of luciferase expression

after butyrate exposure than that observed in cells trans-

fected with the wild-type promoter construct (Fig. 4, P <

0.05). These data confirm that the canonical CRE element is

directly involved in the transcriptional activation of the TH

gene promoter by butyrate.

4. Discussion

In this report, we show that the proximal promoter of

the rat TH gene (�773/+27 bp) contains sufficient

genetic information to confer butyrate responsiveness to

a reporter gene. Our data identified two regions of the

TH promoter that are important for enabling butyrate-

dependent responses. One region involves the canonical

CRE site (TGACGTCA; �45 to �38 bp upstream of the

TH start site). The other region involves a butyrate

response element �509 to �504 bp upstream of the TH

start site (GCCTGG), a sequence originally characterized

in the ppEnk gene in approximately the same position

relative to the CRE [31]. In addition, we showed that

PC12 cells contain nuclear proteins that can bind to the

TH promoter at the BRE site, that the BRE-bound

protein(s) were competed away by the CRE element or

aCREB antibodies and that the intact CRE element itself

was required for full expression of butyrate-dependent

effects. Since the distal BRE regulatory element of the

TH promoter is ¨500 bp upstream from the TH

transcription start site and both it and the canonical

CRE are required for maximal butyrate-induced expres-

sion, the results suggest folding of the promoter to allow

approximation of the transcription factors bound at both

the CRE and BRE elements. Cross-linking studies and

characterization of bound factors will be needed to

confirm this.

Our previous work showed that both cAMP and MAP

kinase second messenger systems converge on the CREB

Fig. 5. Putative model to explain activation of TH gene transcription by butyrate. In

interaction of CREB and CREB-related transcription factors with the proximal C

formation between butyrate-inducible transcription factors and the distal BRE m

CRE-binding proteins to achieve maximal transcriptional effects.

protein (Shah et al., and DeCastro et al., unpublished results)

and are directly involved in butyrate-induced activation of

TH gene expression. Similarly, other investigators have

shown that activation of PKA and ERK signal transduction

systems occurs in response to butyrate exposure in colonic

epithelial and other cell lines [1,27]. Although these

comparative studies are intriguing, the specific upstream

activators of the cAMP cascade induced by butyrate in PC12

cells remain to be fully elucidated. However, SCFAs like

butyrate were recently identified as specific agonists for

orphan G-protein-coupled receptors (GPCRs). More specif-

ically, SCFAs were shown to act through GPR41 and GPR43

in leukocytes, were coupled to IP3 formation, caused

intracellular Ca2+ release and activated ERK1/2 and the

cAMP cascade [7,33,44]. Whether similar receptors are

expressed in PC12 cells or mediate the transcriptional effects

of butyrate on neurotransmitter-related genes is currently

under investigation.

Since alteration in the TH-BRE sequence changes

binding of the nuclear proteins at this site as well as

plasmid function, we conclude that the binding of proteins

to this TH promoter segment is sequence-specific. Binding

of nuclear proteins at the BRE element is competed away

by the CRE sequence (or aCREB antibodies), suggesting

that bound proteins are either CREB or CREB-related

proteins. The significant decrease in reporter gene activity

in response to butyrate after a single point mutation at

either the BRE or CRE sequence provides additional

evidence that both elements are functional with regard to

response to butyrate. Taken together, the butyrate data

indicate that both the upstream BRE and downstream CRE

elements interact.

It is of interest that valproic acid is a structural dimmer of

the CH3CH2-R functional motif we identified as the active

site of butyrate-induced gene expression [37,42]. Moreover,

in humans, valproate is well recognized for its utility in

treatment of attention deficit disorders and seizures [4,5].

Valproate, like butyrate, can induce TH expression (in the

basal conditions activation of TH gene transcription depends largely on the

RE motif in the promoter. Stimulation with butyrate increases the complex

otif, which may directly or indirectly (through co-activators) interact with

P. Patel et al. / Developmental Brain Research 160 (2005) 53–6260

locus coeruleus), and both are known to inhibit histone

deacetylase [18,26]. The current work expands upon our

previous observations and offers a compelling clinical

consideration that dual CH3CH2-R functional motifs of

valproate may use mechanisms similar to those we

elucidated herein for butyrate.

Previous reports indicate that most of the effects of

butyrate utilize one of three categorical cellular or molecular

processes: (i) inhibition of histone deacetylase and attendant

chromatin remodeling, (ii) induction of cis- and trans-acting

butyrate-dependent transcription factors affecting specific

genes and (iii) regulation of the turnover of specific mRNAs

by butyrate response factors [20,36,38,49]. Our studies

utilizing the PC12 model system have primarily focused

on butyrate in transcriptional control mechanisms. However,

none of the experiments to date can entirely exclude the

additional possibility that inhibition of histone deacetylation

or mRNA stabilization may contribute to changes in the

accumulation of mRNA of the endogenous TH gene.

Nevertheless, in the current series of experiments, since we

have used two different TH promoter-reporter constructs

(CAT and luciferase), TH mRNA stabilization mechanisms

appear unlikely unless all mRNAs are stabilized as a

generalized phenomenon after exposure to butyrate, a remote

possibility that, nevertheless, will need to be formally tested.

We of course can also not exclude trans-activation by

other butyrate-induced genes (e.g. transcription factors)

whose expression is simultaneously altered by changes in

the cells’ histone acetylation state due to butyrate exposure.

This form of control would be indirect but could involve

changing expression of relevant transcription factors influ-

encing the TH gene. In support of a direct effect on

transcriptional control mechanisms, our preliminary data

using run-on assays show that TH transcription itself is

directly induced by butyrate.

The biologic and clinical significance of these findings is

of interest from several perspectives. It is well recognized

that maturation of the sympathoadrenal transmitter system

begins during intrauterine life and continues well beyond

infancy [21,48]. In fact, catecholamine release as well as

biosynthesis accelerates after birth, showing a delayed surge

at 7–10 days postnatal age [21]. Concurrent maturation of

synapse formation and the hypothalamic–pituitary–adrenal

cortical axis occurs over this same postnatal time period as

does acquisition of gut flora, fermentation of gut carbohy-

drates (primarily from milk) and absorption of SCFAs into

the blood stream. The SCFAs absorbed include acetate,

propionate, butyrate, valerate and caproate [51]. However, it

is only butyrate and propionate that have substantial gene

regulatory effects [37,42], where butyrate is more potent.

These results allow us to speculate that newborn animals

may, at least in part, have a selective survival advantage

arising from the synergistic interaction between cholinergic

pathways and SCFAs acquired after birth through augmen-

tation of catecholamine biosynthesis, enabling improved

catecholamine-mediated adaptive responses to stress [37,42].

Moreover, a cholinergic–SCFA mechanism appears likely

to be involved with the down-regulation of TH mRNA

levels when high levels of nicotine and butyrate interact

[25]. This particular effect may prove relevant to the loss of

epinephrine release following recurrent hypoglycemic

ketotic stress in the hypoglycemic unawareness syndrome

at any postnatal age [9]. Lastly, the developmental impact of

our findings may also begin to explain the nature of the

teratogenic effects of valproate when it is used clinically

during pregnancy [43].

In summary, using single point mutation methodology,

we show a direct involvement of butyrate-induced, TH

promoter–nuclear protein interactions using EMSA and TH

promoter-reporter gene constructs to assess function. The

data support the coordinate involvement of an upstream

BRE (GCCTGG; �509 to �504 bp of the TH start site) with

a downstream TH canonical CRE element (TGACGTCA;

�45 to�38 bp). These data are consistent with folding of the

TH promoter to bring these two regions into approximation

(see proposed model, Fig. 5).

Acknowledgments

This work was supported by Mead Johnson Nutri-

tionals, institutional grants from the Children’s Foundation

of the Department of Pediatrics, NYMC and by the New

York Medical College Research Endowment Fund under

the New York Medical College Intramural Research Support

Program.

References

[1] H.M. Aukema, L.A. Davidson, B.C. Pence, Y.H. Jiang, J.R. Lupton,

R.S. Chapkin, Butyrate alters activity of specific cAMP-receptor

proteins in a transgenic mouse colonic cell line, J. Nutr. 127 (1997)

18–24.

[2] J.A. Barnard, J.A. Delzell, N.M. Bulus, Short chain fatty acid

regulation of intestinal gene expression, Adv. Exp. Med. Biol. 422

(1997) 137–144.

[3] I.B. Black, J.E. Adler, C.F. Dreyfus, G.M. Jonakait, D.M. Katz, E.F.

LaGamma, K.M. Markey, Neurotransmitter plasticity at the molecular

level, Science 225 (1984) 1266–1270.

[4] R.A. Blaheta, J. Cinatl Jr., Anti-tumor mechanisms of valproate: a

novel role for an old drug, Med. Res. Rev. 22 (2002) 492–511.

[5] C.L. Bowden, A.M. Brugger, A.C. Swann, J.R. Calabrese, P.G.

Janicak, F. Petty, S.C. Dilsaver, J.M. Davis, A.J. Rush, J.G. Small, et

al., Efficacy of divalproex vs. lithium and placebo in the treatment

of mania. The Depakote Mania Study Group, JAMA 271 (1994)

918–924.

[6] M.M. Bradford, A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding, Anal. Biochem. 72 (1976) 248–254.

[7] A.J. Brown, S.M. Goldsworthy, A.A. Barnes, M.M. Eilert, L.

Tcheang, D. Daniels, A.I. Muir, M.J. Wigglesworth, I. Kinghorn,

N.J. Fraser, N.B. Pike, J.C. Strum, K.M. Steplewski, P.R. Murdock,

J.C. Holder, F.H. Marshall, P.G. Szekeres, S. Wilson, D.M. Ignar, S.M.

Foord, A. Wise, S.J. Dowell, The Orphan G protein-coupled receptors

GPR41 and GPR43 are activated by propionate and other short chain

carboxylic acids, J. Biol. Chem. 278 (2003) 11312–11319.

P. Patel et al. / Developmental Brain Research 160 (2005) 53–62 61

[8] M. Bugaut, Occurrence, absorption and metabolism of short chain

fatty acids in the digestive tract of mammals, Comp. Biochem.

Physiol., B 86 (1987) 439–472.

[9] P.E. Cryer, Hypoglycemia-associated autonomic failure in diabetes,

Am. J. Physiol.: Endocrinol. Metab. 281 (2001) E1115–E1121.

[10] J.H. Cummings, E.W. Pomare, W.J. Branch, C.P. Naylor, G.T.

Macfarlane, Short chain fatty acids in human large intestine, portal,

hepatic and venous blood, Gut 28 (1987) 1221–1227.

[11] J.D. DeCristofaro, E.F. LaGamma, Neonatal stress: effects of

hypoglycemia and hypoxia on adrenal tyrosine hydroxylase gene

expression, Pediatr. Res. 36 (1994) 719–723.

[12] J.D. Dignam, P.L. Martin, B.S. Shastry, R.G. Roeder, Eukaryotic gene

transcription with purified components, Methods Enzymol. 101 (1983)

582–598.

[13] N. Faucon Biguet, S. Vyas, J. Mallet, In vitro and in vivo regulation of

the expression of the tyrosine hydroxylase gene, J. Physiol. 85 (1991)

105–109.

[14] M.D. Fitch, S.E. Fleming, Metabolism of short-chain fatty acids by rat

colonic mucosa in vivo, Am. J. Physiol. 277 (1999) G31–G40.

[15] S.E. Fleming, S.Y. Choi, M.D. Fitch, Absorption of short-chain fatty

acids from the rat cecum in vivo, J. Nutr. 121 (1991) 1787–1797.

[16] B.P. Fung, S.O. Yoon, D.M. Chikaraishi, Sequences that direct rat

tyrosine hydroxylase gene expression, J. Neurochem. 58 (1992)

2044–2052.

[17] J.G. Glauber, N.J. Wandersee, J.A. Little, G.D. Ginder, 5V-flankingsequences mediate butyrate stimulation of embryonic globin gene

expression in adult erythroid cells, Mol. Cell. Biol. 11 (1991)

4690–4697.

[18] M. Gottlicher, S. Minucci, P. Zhu, O.H. Kramer, A. Schimpf, S.

Giavara, J.P. Sleeman, F. Lo Coco, C. Nervi, P.G. Pelicci, T. Heinzel,

Valproic acid defines a novel class of HDAC inhibitors inducing

differentiation of transformed cells, EMBO J 20 (2001) 6969–6978.

[19] L.A. Greene, A.S. Tischler, Establishment of a noradrenergic clonal

line of rat adrenal pheochromocytoma cells which respond to nerve

growth factor, Proc. Natl. Acad. Sci. U. S. A. 73 (1976) 2424–2428.

[20] M. Grunstein, Histone acetylation in chromatin structure and tran-

scription, Nature 389 (1997) 349–352.

[21] R.W. Hamill, E.F. LaGamma, Autonomic nervous system develop-

ment, in: S.R. B (Ed.), Autonomic Failure: A Textbook of Clinical

Disorders of the Autonomic Nervous System, Oxford Univ. Press,

New York, 1992, pp. 15–35.

[22] B. Hiremagalur, B. Nankova, J. Nitahara, R. Zeman, E.L. Sabban,

Nicotine increases expression of tyrosine hydroxylase gene. Involve-

ment of protein kinase A-mediated pathway, J. Biol. Chem. 268

(1993) 23704–23711.

[23] M. Holloway, E.F.L. Gamma, Enhancement of Forskolin-induced

preproenkephalin (ppEnk) expression in butyrate differentiated PC12

cells maps to a 404 bp upstream element, Pediatr. Res. 30 (4) (1994) A

402.

[24] T. Hoverstad, O. Fausa, A. Bjorneklett, T. Bohmer, Short-chain fatty

acids in the normal human feces, Scand. J. Gastroenterol. 19 (1984)

375–381.

[25] K.E. Inouye, O. Chan, J.T. Yue, S.G. Matthews, M. Vranic, Effects of

diabetes and recurrent hypoglycemia on the regulation of the

sympathoadrenal system and hypothalamo–pituitary–adrenal axis,

Am. J. Physiol.: Endocrinol. Metab. 288 (2005) E422–E429.

[26] M.S. Jansen, S.C. Nagel, P.J. Miranda, E.K. Lobenhofer, C.A. Afshari,

D.P. McDonnell, Short-chain fatty acids enhance nuclear receptor

activity through mitogen-activated protein kinase activation and

histone deacetylase inhibition, Proc. Natl. Acad. Sci. U. S. A. 101

(2004) 7199–7204.

[27] P.R. Kiela, E.R. Hines, J.F. Collins, F.K. Ghishan, Regulation of the

rat NHE3 gene promoter by sodium butyrate, Am. J. Physiol.:

Gastrointest. Liver Physiol. 281 (2001) G947–G956.

[28] K.S. Kim, M.K. Lee, J. Carroll, T.H. Joh, Both the basal and inducible

transcription of the tyrosine hydroxylase gene are dependent upon a

cAMP response element, J. Biol. Chem. 268 (1993) 15689–15695.

[29] H.S. Kim, S.J. Hong, M.S. LeDoux, K.S. Kim, Regulation of the

tyrosine hydroxylase and dopamine beta-hydroxylase genes by the

transcription factor AP-2, J. Neurochem. 76 (2001) 280–294.

[30] S.C. Kumer, K.E. Vrana, Intricate regulation of tyrosine hydroxylase

activity and gene expression, J. Neurochem. 67 (1996) 443–462.

[31] E.F. LaGamma, J. Chua, C.D. Kobasiuk, E.L. Sabban, M.J. Evinger,

Synergistic interaction between bacterial fermentation of dietary

carbohydrate and postnatal maturation of the autonomic nervous

system (ANS): a hypothesis on evolution of two animal kingdoms,

Pediatr. Res. 48 (2000) A752535.

[32] M. Lazaroff, S. Patankar, S.O. Yoon, D.M. Chikaraishi, The cyclic

AMP response element directs tyrosine hydroxylase expression in

catecholaminergic central and peripheral nervous system cell lines

from transgenic mice, J. Biol. Chem. 270 (1995) 21579–21589.

[33] E. Le Poul, C. Loison, S. Struyf, J.Y. Springael, V. Lannoy, M.E.

Decobecq, S. Brezillon, V. Dupriez, G. Vassart, J. Van Damme, M.

Parmentier, M. Detheux, Functional characterization of human

receptors for short chain fatty acids and their role in polymorphonu-

clear cell activation, J. Biol. Chem. 278 (2003) 25481–25489.

[34] E.J. Lewis, C.A. Harrington, D.M. Chikaraishi, Transcriptional

regulation of the tyrosine hydroxylase gene by glucocorticoid and

cyclic AMP, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 3550–3554.

[35] L.J. Lewis-Tuffin, P.G. Quinn, D.M. Chikaraishi, Tyrosine hydrox-

ylase transcription depends primarily on cAMP response element

activity, regardless of the type of inducing stimulus, Mol. Cell.

Neurosci. 25 (2004) 536–547.

[36] K.N. Maclean, I.A. McKay, S.A. Bustin, Differential effects of sodium

butyrate on the transcription of the human TIS11 family of early-

response genes in colorectal cancer cells, Br. J. Biomed. Sci. 55 (1998)

184–191.

[37] P. Mally, R. Mishra, S. Gandhi, M.H. Decastro, B.B. Nankova, E.F.

Lagamma, Stereospecific regulation of tyrosine hydroxylase and

proenkephalin genes by short-chain fatty acids in rat PC12 cells,

Pediatr. Res. 55 (2004) 847–854.

[38] P.A. Marks, V.M. Richon, R.A. Rifkind, Histone deacetylase inhib-

itors: inducers of differentiation or apoptosis of transformed cells,

J. Natl. Cancer Inst. 92 (2000) 1210–1216.

[39] P.G. McCaffrey, D.A. Newsome, E. Fibach, M. Yoshida, M.S. Su,

Induction of gamma-globin by histone deacetylase inhibitors, Blood

90 (1997) 2075–2083.

[40] B. Nankova, R. Kvetnansky, A. McMahon, E. Viskupic, B. Hirema-

galur, G. Frankle, K. Fukuhara, I.J. Kopin, E.L. Sabban, Induction of

tyrosine hydroxylase gene expression by a nonneuronal nonpituitary-

mediated mechanism in immobilization stress, Proc. Natl. Acad. Sci.

U. S. A. 91 (1994) 5937–5941.

[41] B. Nankova, B. Hiremagalur, A. Menezes, R. Zeman, E. Sabban,

Promoter elements and second messenger pathways involved in

transcriptional activation of tyrosine hydroxylase by ionomycin, Brain

Res. Mol. Brain Res. 35 (1996) 164–172.

[42] B.B. Nankova, J. Chua, R. Mishra, C.D. Kobasiuk, E.F. La Gamma,

Nicotinic induction of preproenkephalin and tyrosine hydroxylase

gene expression in butyrate-differentiated rat PC12 cells: a model for

adaptation to gut-derived environmental signals, Pediatr. Res. 53

(2003) 113–118.

[43] H. Nau, R.S. Hauck, K. Ehlers, Valproic acid-induced neural tube

defects in mouse and human: aspects of chirality, alternative drug

development, pharmacokinetics and possible mechanisms, Pharmacol.

Toxicol. 69 (1991) 310–321.

[44] N.E. Nilsson, K. Kotarsky, C. Owman, B. Olde, Identification of a free

fatty acid receptor, FFA2R, expressed on leukocytes and activated by

short-chain fatty acids, Biochem. Biophys. Res. Commun. 303 (2003)

1047–1052.

[45] N.A. Papanikolaou, E.L. Sabban, Ability of Egr1 to activate tyrosine

hydroxylase transcription in PC12 cells. Cross-talk with AP-1 factors,

J. Biol. Chem. 275 (2000) 26683–26689.

[46] E.L. Sabban, Control of tyrosine hydroxylase gene expression in

chromaffin and PC12 cells, Semin. Cell Dev. Biol. 8 (1997) 101–111.

P. Patel et al. / Developmental Brain Research 160 (2005) 53–6262

[47] B. Seed, J.Y. Sheen, A simple phase-extraction assay for chloramphe-

nicol acyltransferase activity, Gene 67 (1988) 271–277.

[48] T. Slotkin, Development of the Sympathoadrenal Axis, Humana Press,

New York, 1985, pp. 69–96.

[49] G. Stoecklin, M. Colombi, I. Raineri, S. Leuenberger, M. Mallaun, M.

Schmidlin, B. Gross, M. Lu, T. Kitamura, C. Moroni, Functional

cloning of BRF1, a regulator of ARE-dependent mRNA turnover,

EMBO J. 21 (2002) 4709–4718.

[50] O. Szylit, C. Maurage, P. Gasqui, F. Popot, A. Favre, F. Gold, J.

Borderon, Fecal short-chain fatty acids predict digestive disorders in

premature infants, J. Parenter. Enteral. Nutr. 22 (1998) 136–141.

[51] D.L. Topping, P.M. Clifton, Short-chain fatty acids and human colonic

function: roles of resistant starch and nonstarch polysaccharides,

Physiol. Rev. 81 (2001) 1031–1064.

[52] C.H. Wang, Y.P. Tsao, H.J. Chen, H.L. Chen, H.W. Wang, S.L.

Chen, Transcriptional repression of p21((Waf1/Cip1/Sdi1)) gene by

c-jun through Sp1 site, Biochem. Biophys. Res. Commun. 270

(2000) 303–310.

[53] G. Weisinger, The transcriptional regulation of the preproenkephalin

gene, Biochem. J. 307 (1995) 617–629.

[54] C. Yang, H.S. Kim, H. Seo, K.S. Kim, Identification and character-

ization of potential cis-regulatory elements governing transcriptional

activation of the rat tyrosine hydroxylase gene, J. Neurochem. 71

(1998) 1358–1368.

[55] J. Yang, Y. Kawai, R.W. Hanson, I.J. Arinze, Sodium butyrate induces

transcription from the G alpha(i2) gene promoter through multiple Sp1

sites in the promoter and by activating the MEK-ERK signal

transduction pathway, J. Biol. Chem. 276 (2001) 25742–25752.