Download Widespread Organ Expression of the Rat Proenkephalin Gene

Widespread Organ Expression of the
Rat Proenkephalin Gene during Early
Postnatal Development
David Kew and Daniel L. Kilpatrick
Neurobiology Group
Worcester Foundation for Experimental Biology
Shrewsbury, Massachussetts 01545
RESULTS
The opioid peptides have been implicated as potential regulators of cell development in nervous and
reproductive tissues. A survey of proenkephalin
gene expression during rat development showed
that the mRNA for this opioid precursor is present at
substantial concentrations in several developing tissues (kidney, liver, skin, skeletal muscle, and lung)
that have essentially undetectable levels in adults.
In neonatal rats, skeletal muscle has greater concentrations of this transcript than brain. Polysomal
analysis further demonstrated that proenkephalin
mRNA is actively translated in skeletal muscle from
newborn rats. These results raise the possibility that
proenkephalin and its products perform a general
regulatory role in cell proliferation or differentiation.
(Molecular Endocrinology 4: 337-340, 1990)
Proenkephalin mRNA in Various Tissues of
Immature and Adult Rats
A comparison of the relative abundances of proenkephalin mRNA was initially made between tissues from
14-day-old and adult (90- to 115-day-old) rats. In both
age groups the concentrations of this transcript were
relatively high in brain and heart, but were below the
limits of detection in kidney, liver, and spleen (Fig. 1;
see also Fig. 2). In contrast, proenkephalin mRNA was
readily detectable in skeletal muscle and lung from 14day-old rats, but was either markedly reduced in concentration (lung) or undetectable (muscle) in the adult
organs. The size of this transcript was the same as that
observed in brain and other adult somatic tissues [1450
nucleotides (8)].
Speculating that expression of the proenkephalin
gene may be correlated with rapid growth or differentiation, we then assayed the organs of neonatal rats (1-2
days of age). The results demonstrated that the proenkephalin gene was more widely expressed than at the
older ages (Fig. 2). In addition to neonatal brain, heart,
and lung, proenkephalin mRNA was also observed in
newborn skeletal muscle, liver, kidney, skin, and intestine, tissues that have negligible or undetectable transcript levels in the adult (see Fig. 1). The only organ
from neonatal rat pups examined in this survey that did
not contain detectable amounts of proenkephalin
mRNA was the spleen (Fig. 2).
It should be noted that the abundance of proenkephalin mRNA observed in developing nonneural tissues
is quite substantial. For example, expression in neonatal
skeletal muscle is greater than that observed in neonatal brain (Fig. 2). Transcript abundance in muscle
shows a steady several-fold decline during the first 2
weeks after birth and falls below the limits of detection
in the adult (Fig. 3). In contrast, both brain and heart
exhibited the opposite pattern of developmental expression, with proenkephalin mRNA levels being substantially higher in the adult tissues (Fig. 2).
INTRODUCTION
Peptide growth factors play a major role in the control
of cell development. Recent studies have suggested
that peptides typically associated with neural and/or
endocrine functions also have growth-promoting effects
and may be involved in tumorigenesis (1, 2). Included
in this group are opioid peptides such as /3-endorphin
and the enkephalins. /S-Endorphin has modulatory effects on the proliferation of lymphocytes and cell lines
derived from small cell carcinoma of the lung (2, 3).
Opioid peptides derived from proenkephalin have been
implicated as regulators in the early development of
neuronal and glial cells (4,5). Further evidence suggests
that proenkephalin and its products may play a more
general role in tissue development. For example, the
proenkephalin gene is expressed by testicular somatic
cells during postnatal development (6) and by developing spermatogenic cells in the adult rat (7). We, therefore, sought to determine the pattern of developmental
expression for the proenkephalin gene in different rat
organs and tissues. Our results are consistent with the
hypothesis that proenkephalin-derived peptides are involved in cell development in a variety of tissues.
Polysome Distribution of Proenkephalin mRNA in
Developing Muscle
0888-8809/90/0337-0340S02.00/0
Molecular Endocrinology
Copyright © 1990 by The Endocrine Society
An important question arising from the foregoing data
is whether proenkephalin mRNA present exclusively
337
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MOL ENDO-1990
338
during development is functional, i.e. is translated into
proenkephalin. This was addressed by examining the
polysome distribution of this transcript in neonatal rat
skeletal muscle, which has relatively high concentrations of proenkephalin mRNA at this age. This approach
was employed instead of peptide quantitation since
several tissues that contain and actively translate
proenkephalin mRNA do not exhibit significant concentrations of proenkephalin products, apparently due to a
rapid rate of peptide secretion or turnover (6, 9).
Proenkephalin mRNA was localized to the polysomal
fractions in gradients prepared from neonatal skeletal
muscle (Fig. 4). This polysome profile is comparable to
that observed for other tissues previously shown to
efficiently translate proenkephalin mRNA, such as brain
(6). Proenkephalin transcripts were released from polysomes in the presence of EDTA (Fig. 4), indicative of
specific polysomal association. This transcript is, therefore, actively translated during postnatal muscle development. Although not specifically examined here, it is
likely that proenkephalin mRNA is also efficiently translated in other developing organs in which it is present.
-I8S
Fig. 2. Comparison of Proenkephalin mRNA in Neonatal (neo.)
and Adult (ad.) Rat Tissues
Each lane contained 20 ng poly(A)+ RNA. A, Neonatal tissues and two adult tissue controls. B, Direct comparison of
neonatal and adult brain RNAs. This is a shorter exposure
than in A.
DISCUSSION
The results reported here indicate that the opioid peptide precursor proenkephalin is produced by a variety
of rat tissues during early postnatal development. This
group includes tissues derived from embryonic endoderm (liver and lung), mesoderm (skeletal muscle, heart,
and kidney), and ectoderm (brain). Skin has both ectodermal (epidermis) and mesodermal components (dermis), and intestine has both endodermal and mesodermal origins, the relative contributions of which were
not determined here. In most cases, the level of proen-
-I8S
Fig. 1. RNA Gel-Blot Analysis of Proenkephalin mRNA in
Several Adult and Juvenile (14-Day-Old) Rat Tissues
Each tissue is represented by 30 ^g poly(A)+ RNA. Samples
were prepared and analyzed as described in Materials and
Methods. A, Fourteen-day-old tissues. B, Adult tissues.
t-
-I8S
Fig. 3. Age-Dependent Changes in the Abundance of Proenkephalin mRNA in Rat Skeletal Muscle
Each lane contained 20 ng poly(A)+ RNA. RNA gel-blot
analysis was performed as outlined in Materials and Methods.
kephalin gene expression is markedly reduced or undetectable in the adult rat. Two exceptions to this
pattern are brain and heart, which exhibit higher proenkephalin mRNA abundance in the adult. This probably
reflects mRNA production by cells programed to express the proenkephalin gene at relatively high levels in
the mature animal. It, therefore, appears that in addition
to its production by certain fully differentiated tissues
(e.g. brain, adrenal medulla, heart, and reproductive
organs), proenkephalin expression is also associated
with cell proliferation or differentiation. The absence of
proenkephalin mRNA in postnatal rat spleen may indicate that this association is not general, although perhaps developmental expression of proenkephalin in this
organ is limited to the fetal period, which was not
examined here. More extensive analysis, using in situ
hybridization, will be necessary to further identify and
characterize the proenkephalin-expressing cells and
their fates during the development of different tissues.
339
Developmental Expression of Proenkephalin
HKMG
-18 S
HKE
I
top
-18 S
8
9
10
II
12
bottom
Fig. 4. Polysome Profile of Proenkephalin mRNA in Neonatal Rat Skeletal Muscle
Polysomes are intact in sucrose gradients containing HKMG buffer and are dissociated in HKE gradients (containing EDTA in
place of Mg2+). The preparation and analysis of postmitochondrial supernates are described in Materials and Methods. The direction
of sedimentation is from left to right as shown. Based on the absorbance profile at 260 nm (not shown), polysomes were localized
to fractions 7-12, and the postpolysomal region was found in fractions 1-3.
The recent demonstration of proenkephalin expression by developing astrocytes and neurons (4, 5, 10)
suggests that the peptide products derived from this
precursor may regulate cell differentiation or proliferation within the brain. Growth-promoting effects of enkephalins and other opioid peptides on cultured neurons and glial cells have been reported (11,12). The
much wider tissue distribution of proenkephalin gene
expression during early postnatal development and its
subsequent decline with age shown here are suggestive
of a more general association with cell growth. Proenkephalin gene expression in immature testes and in
developing male germ cells is consistent with this notion. As pointed out above, the abundance of proenkephalin mRNA in certain nonneural tissues (e.g. skeletal muscle) is even greater than that observed in brain
at early stages of development.
Bombesin-like peptides are selectively produced in
lung during development and have been shown to
stimulate cell proliferation (2). Other neuropeptides that
have been implicated as growth regulators include vasopressin, gastrin, the neuromedins (substance-P and
substance-K), and /3-endorphin (2). Proenkephalin products, therefore, may be part of a class of peptides with
both growth-related activities during development as
well as neuro/endocrine functions in fully differentiated
tissues. This apparent duality of peptide function may
have evolved long ago, as indicated by the neurotransmitter and mitogenic/differentiative activities exhibited
by the head activator peptide in the coelenterate Hydra
(13).
The work reported here generally supports the conclusions of previous immunohistochemical analyses of
enkephalin-like immunoreactivity in developing rat tissues, with at least one exception. Zagon et al. (14)
observed substantial immunoreactivity in immature rat
spleen, while we found no proenkephalin mRNA. This
underlines the necessity for biochemical characterization of detected immunoreactivity, which was not performed in these earlier studies. The relationship of this
enkephalin-like material to authentic enkephalin or en-
kephalin-containing peptides derived from proenkephalin, therefore, remains uncertain. An understanding
of the possible role of proenkephalin in tissue development will ultimately follow determination of the specific
peptide products synthesized and secreted by developing cells.
Another intriguing question raised by these studies
concerns the mechanism(s) responsible for the decline
in proenkephalin mRNA abundance with age. One possibility is that this reflects development-dependent
repression of proenkephalin gene transcription, as described for a-fetoprotein (15). Posttranscriptional processes (e.g. mRNA destabilization) also may contribute.
Gene transfer will be a valuable approach in examining
these possibilities.
MATERIALS AND METHODS
Animals and Tissues
Adult and newborn CD-1 rats (Charles River Laboratories,
Wilmington, MA) were killed by CO2 inhalation, and tissues
were either extracted immediately or frozen on dry ice and
stored at - 8 0 C for later analysis. The skeletal muscle used
was from the thigh, shoulder, and upper forelimb.
Tissue Comparisons
Fresh or frozen tissues were weighed and homogenized in
approximately 10 vol guanidine thiocyanate buffer with a Tekmar homogenizer, and the RNA was collected by centrifuging
through CsCI buffer (16). Poly(A)+ RNA was selected with
oligo-dT (Pharmacia, Piscataway, NJ). Equal amounts of
poly(A)+ RNA were separated on formaldehyde-agarose gels,
and blotted to Gene-Screen Plus membranes (New England
Nuclear, Boston, MA). Blots were probed with cDNA to rat
proenkephalin [pRPE-1 (165-600)] (8) labeled by random priming with [32P]dCTP. Blots and probe were incubated overnight
at 42 C, with final washes at 56 C in 15 mM NaCI-1.5 ITIM
sodium citrate-0.1% sodium dodecyl sulfate (17,18). Analyses
were performed on at least two different RNA preparations in
each case.
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MOL ENDO-1990
340
Polysome Analysis
The protocol was based on that of Kleene et al. (19). Fresh
newborn skeletal muscle was homogenized in a motorized
glass/Teflon homogenizer at 4 C in 4 vol HKMG buffer (20 miui
HEPES-100 mM KCI-20 miui MgCI2-10 mM EGTA, pH 7.6)
containing 0.5% (wt/vol) Triton X-100-3 ITIM mercaptoethanol200 U/ml RNAsin (Promega, Madison, Wl). Tissue for the
nonspecificity control was homogenized in HK buffer (20 mM
HEPES-100 mM KCI, pH 7.6), also containing Triton X-100,
mercaptoethanol, and RNAsin. Homogenates were centrifuged to pellet nuclei and debris, then 0.5 M EDTA (pH 7.6)
was added to the HK sample to a final concentration of 20 mM
(HKE buffer) to dissociate the polysomes. The supemates
were overlaid onto linear gradients of 10-40% (wt/wt) sucrose
with 60% sucrose cushions, made up in HKE or HKMG buffer
as appropriate. Gradients were spun in a Beckman SW28
rotor (Beckman, Palo Alto, CA) for 2.5 h at 28,000 rpm at 4
C, and decelerated without the brake.
Gradient fractions were diluted with an equal volume of 0.4
M sodium acetate (pH 5.5), mixed with 50 (ig yeast tRNA and
20 ng glycogen carrier, and precipitated at - 2 0 C overnight
with isopropanol. RNA was extracted and purified further by
the method of Chomczynski and Sacchi (20). Fractions were
dissolved in guanidine thiocyanate buffer, extracted with
phenol-chloroform-isoamyl alcohol, and again precipitated with
isopropanol. Precipitated fractions were dissolved in 0.5%
sodium dodecyl sulfate and reprecipitated with ethanol. Equivalent percentages of each fraction were run on a gel, blotted,
and probed for proenkephalin mRNA as described above.
Note Added in Proof
Two additional reports describing the developmental
expression of proenkaphalin have recently appeared:
Keshet E, Polakiewicz RD, Itin A, Orney A, Rosen H 1989
Proenkephalin A is expressed in mesodermal lineages during
organogenesis. EMBO J 8:2917-2923;
Springhom JP, Claycomb WC 1989 Proenkephalin mRNA
expression in developing rat heart and in cultured ventricular
cardiac muscle cells. Biochem J 258:73-78.
Acknowledgments
We thank Dr. Steven Zinn and Mr. Carl Schell for their help.
Received October 9, 1989. Revision received November
14,1989. Accepted November 16,1989.
Address requests for reprints to: Dr. Daniel L. Kilpatrick,
Worcester Foundation for Experimental Biology, Neurobiology
Group, Shrewsbury, Massachussetts 01545.
This work was supported by NIH Grants DK-35855 and DK36486 (to D.L.K.) and grants to the Worcester Foundation
from the Andrew W. Mellon Foundation and the Edward John
Noble Foundation.
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