1 Department of Pharmaceutical Sciences and University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2 Jackson Laboratory, Bar Harbor, Maine 04609
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ABSTRACT |
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Strain A/J mice, which
are predisposed to experimentally induced asthma and adenocarcinoma,
have the lowest pulmonary protein kinase (PK) C activity and content
among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to
that in other strains, so this difference reflects strain-dependent
postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than
in A/J lungs, and the protein and mRNA concentrations of PKC-, the
major pulmonary PKC isozyme, are two- to threefold higher in B6 mice.
These differences result from more than a single gene as assessed in
F1, F2, and backcross progeny of B6 and A/J
parents. Quantitative trait locus (QTL) analysis of 23 A×B and B×A
recombinant inbred strains derived from B6 and A/J progenitors
indicates a major locus regulating lung PKC-
content that maps near
the Pkc
structural gene on chromosome 11 (D11MIT333; likelihood ratio statistic = 12.5) and a
major locus controlling PKC activity that maps on chromosome 3 (D3MIT19; likelihood ratio statistic = 15.4). The
chromosome 11 QTL responsible for low PKC-
content falls within QTLs
for susceptibilities to lung tumorigenesis and ozone-induced toxicity.
protein kinase C; inbred mouse strains; developmental regulation; genetic mapping; lung tumor susceptibility
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INTRODUCTION |
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STRAIN A/J mice are more susceptible than other strains to pulmonary damage including lung tumorigenesis (21) and ozone-induced lung injury (31). For example, nearly 100% of A/J mice develop one or a few lung adenomas spontaneously by the end of their lifetime and several dozen adenomas within a few months after injection of a carcinogen such as urethan. In contrast, no tumors appear in untreated C57BL/6J (B6) mice, and less than half of the B6 mice injected with urethan develop even a single tumor. Strain-dependent lung tumor susceptibilities display a continuum ranging from the highly sensitive A/J strain to resistant strains such as B6. At least 14 genetic loci, variously designated as Pas (pulmonary adenoma susceptibility), Par (pulmonary adenoma resistance), and Sluc (susceptibility to lung cancer), have been assigned to chromosomal locations (20). The mapping techniques used include recombinant inbred (RI) (29), quantitative trait locus (QTL) (12), and recombinant congenic strains (11). With the exception of the K-ras protooncogene as Pas1 (17), the identities of these genes have not been firmly established. Identifying gene products that confer resistance to lung cancer would provide new tools with the potential for chemoprevention and therapy.
The pulmonary content and activity of protein kinase (PK) C may
contribute to these strain differences in susceptibility to lung
tumorigenesis. A proposed candidate for Par3, a pulmonary adenoma resistance gene on chromosome 18, is the PKC isozyme PKC- (29). PKC regulates pulmonary differentiation and is also
associated with proliferative quiescence. PKC activation causes
immortalized, nontumorigenic lung epithelial cells to round up
(28), a morphological change that is facilitated by
altered cytoskeletal architecture (7) and terminated by
calpain-mediated proteolysis of PKC-
(6). PKC activity
increases when these cultured lung cells undergo density-dependent
growth inhibition (4), and PKC-
content decreases on
neoplastic transformation (6). During the compensatory
hyperplasia that repairs chemically damaged lungs, both PKC activity
(23) and PKC-
content (26) decrease.
Thus it is of great interest that A/J lungs have less PKC activity (22) and content (24) than lungs from other inbred strains of mice. These variations reflect a developmental difference because the pulmonary PKC concentration and activity in A/J mice were similar at birth to those in BALB/cBy (CBy) mice, but CBy PKC content and activity increased postnatally to a greater extent than those in A/J mice (24). This difference was observed in isolated Clara cells as well as in whole lung extracts (4). The Clara cells and alveolar type 2 pneumocytes, stem cells of their respective compartments (8, 9), are the probable precursor cells of mouse lung tumors (19). Consistent with this, the basal rates of Clara and type 2 cell proliferation in various strains reflect their relative lung tumor susceptibilities (35).
Herein we describe the following: 1) of 22 inbred strains,
only adult A/J mice exhibit both low PKC activity and content in the
lung; 2) PKC- is the isozyme responsible for this strain difference in PKC content; 3) PKC-
protein variation is a
consequence of differential PKC-
mRNA concentrations; 4)
standard Mendelian crosses between A/J and B6 mice suggest that
multiple genes determine this difference; and 5) two loci
consisting of a major locus that regulates PKC-
content on
chromosome 11 near the PKC-
structural gene and a locus on
chromosome 3 that affects PKC activity were identified by QTL analysis
of A×B and B×A RI strains. QTLs for susceptibility to lung
tumorigenesis (10) and ozone-induced lung toxicity
(31) have been reported at the chromosome 11 Pkc
site.
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METHODS |
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Materials.
A/J, B6, A×B and B×A RI strains, CBy, NZW/J, LP/J, P/J, PL/J,
CE/J, SM/J, 129/J, RF/J, MOLD/RKIII, RIII/SmMOB, CAST/Ei, DBA/2J, YBR,
SK, CXB 3, CXB 4, and Mus spretus mice were purchased from Jackson Laboratory (Bar Harbor, ME). GRS/N mice were obtained from the
National Institutes of Health (Bethesda, MD). SS/N mice were a generous
gift from Dr. Susan Fischer (University of Texas, Smithville, TX).
AB6F1, AB6F2, (A×B6)F1 × A,
and (A×B6)F1 × B6 backcross mice were bred in our
facility at the University of Colorado (Denver). Mice were maintained
on aspen chip hardwood bedding (Northeastern Products, Warrenburg, NY),
fed Wayne Rodent Blox (Teklad Premier, Madison, WI) and tap water ad
libitum, and kept on a 12:12-h light-dark cycle. Pulmonary PKC
activities are maximal by 6 wk of age (22). Male and
female mice 6-14 wk of age were used interchangeably because no
gender differences in PKC activity were observed. Pan-PKC antibody was
kindly provided by Dr. Curtis Ashendale (Purdue University, West
Lafayette, IN). PKC- antibody was purchased from Upstate
Biotechnology (Lake Placid, NY), and PKC-
, PKC-
, PKC-
,
PKC-
, and PKC-
antibodies and alkaline phosphatase-conjugated
anti-mouse IgG and anti-rabbit IgG secondary antibodies were from Life
Technologies (Gaithersburg, MD). Immobilon-P polyvinylidene
difluoride membranes were purchased from Millipore (Bedford,
MA), and Chemilume 490 was from Lumigen (Detroit, MI). Bovine brain
PKC-
cDNA (pbPKC-
21), encompassing nucleotides 590-1120 of
the published sequence (encodes the calcium and ATP binding sites), was
purchased from American Type Culture Collection (Manassas, VA). All
other chemicals were purchased from Sigma (St. Louis, MO).
Sample preparation.
Lungs were perfused as previously described (25) and
solubilized in homogenization buffer [20 mM HEPES (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol, and 10% (vol/vol) glycerol]
containing freshly made protease inhibitors (5 µg/ml of aprotinin, 10 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Soluble
(cytosolic) and particulate fractions were separated by a 30-min,
16,000 g centrifugation and immediately mixed (1:1) with 2×
gel sample buffer [0.1 M, Tris-Cl (pH 6.8), 1% (wt/vol) sodium
dodecyl sulfate (SDS), 2% (vol/vol) -mercaptoethanol, 20%
(vol/vol) glycerol, and 0.025% (wt/vol) pyronine Y]. An aliquot of
each sample was saved for protein quantification (18).
PKC protein content and activity.
Pan-PKC Western blot analysis and PKC activity assays were performed as
previously described (25). The pan-PKC antibody recognizes
PKC-, PKC-
, and PKC-
. Because we could not detect PKC-
or
PKC-
protein, the results obtained with this antibody correspond to
those found with the PKC-
-specific antibody. Activity assays that
measure the phosphorylation of histone III by
[
-32P]ATP in the presence and absence of
phosphatidylserine were performed as previously described
(22). The histone substrate does not distinguish among PKC isozymes.
PKC isozyme quantitation.
Equal amounts of cytosolic protein (100 µg/lane) were applied to
7.5% SDS-PAGE. Separated proteins were electroblotted onto Immobilon-P
membrane, and detection of PKC-, PKC-
, and PKC-
with specific
antibodies was performed as previously described (6).
PKC-
bands were quantified by densitometry; A/J and B6 lung samples
were run on each gel to serve as samples for normalization.
Northern blot analysis.
Lungs were harvested from 6- to 8-wk-old A/J and B6 mice, and total RNA
was prepared with the RNeasy kit (QIAGEN, Valencia, CA). Before the
cell lysate was loaded onto the spin column, debris was removed by
centrifugation in a microfuge at 16,000 g for 5 min. Where
applicable, poly(A)-selected RNA was prepared from total RNA with the
Oligotex mRNA Mini Kit (QIAGEN). For each sample, 100 µg of total RNA
were used as starting material in isolating poly(A) RNA. Northern blots
were performed as previously described (36). The blots
were probed with a 531-bp EcoR I cDNA fragment of bovine
brain PKC- (pbPKC-
21) labeled with [
-32P]dCTP to
a specific activity of 108
counts · min
1 · µg
1 with
the RadPrime kit (GIBCO BRL, Life Technologies, Gaithersburg, MD). The
blots were hybridized overnight at 60°C in 15 ml of prehybridization buffer containing 50-75 ng of denatured labeled probe, washed twice at 60°C in a solution of 2× saline-sodium citrate (SSC; 0.15 M
NaCl and 15 mM sodium citrate)-0.1% SDS for 30 min, once in 1×
SSC-0.1% SDS for 30 min, and once in 0.2× SSC-0.1% SDS for 15 min.
Blots were exposed to X-OMAT AR film with an enhancing screen, and the
results were quantified by densitometry and with a phosphorimager.
RI mapping.
The quantitative traits, lung PKC- content and PKC activity, were
mapped with Map Manager QT version 27 and the database for typed loci
named A×B and B×A QT (35a). This database has been edited to remove
errors, extinct strains, questionable loci, sister strains, and loci
with identical strain distribution patterns that interfere with
permutation tests of significance (34). To determine
whether the likelihood ratio statistics (LRSs) were suggestive,
significant, or highly significant, 1,000 permutations were performed.
The logarithm of odds (LOD) score can be approximated by dividing the
LRS by 4.6.
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RESULTS |
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Strain variations in PKC.
PKC protein in A/J and B6 lung tissue extracts was quantified in 16 independent analyses by subjecting autoradiograms (pan-PKC antibody)
and chemiluminescence films (PKC--specific antibody) to
densitometric analysis. Adult B6 lungs had 2.5-fold more PKC-
protein than adult A/J lungs, and B6 mice had 1.6-fold more PKC activity than A/J mice. Twenty other strains were surveyed for their
pulmonary PKC activity and contents (Table
1). Only A/J mice had both low PKC
activity and content; most strains exhibited both higher PKC
activity and content than A/J mice. The correlation between PKC
activity and PKC-
content in these strains was not significant
(r2 = 0.11; P > 0.05).
Particulate fractions prepared from A/J and B6 mouse lung tissues
reflected the same relative difference in PKC activity and content
(when detectable), but there was much less PKC in the particulate
fraction; hence, we focused on soluble PKC.
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Pulmonary PKC isozyme protein content in A/J and B6 mice.
A/J and B6 pulmonary PKC isozyme distributions were determined. Our
laboratory previously established that the major isozymes in
whole CBy lung extracts (26) and in lung epithelial cell lines derived from A/J, CBy, BALB/c, and Swiss-Webster mice
(6) were PKC-, PKC-
, and PKC-
. These proteins
were indeed detected in soluble and particulate fractions from A/J and
B6 lung extracts (Fig. 1), whereas
PKC-
, PKC-
, and PKC-
were not (data not shown). The multiple
bands in the immunoblots for both PKC-
and PKC-
may reflect
differentially phosphorylated species or are a consequence of limited
proteolysis. More PKC-
was contained in B6 lungs than in A/J lungs,
whereas no strain differences were detected in PKC-
and PKC-
protein contents.
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Pulmonary PKC- mRNA content in A/J and B6 mice.
To determine whether these differences in protein expression reflected
differential mRNA contents, Northern blot analysis was performed on RNA
samples prepared from A/J and B6 mouse lungs. Long exposure of film
showed threefold more PKC-
mRNA in B6 than in A/J lungs; this was
true whether total RNA or poly(A)-enriched mRNA fractions were used
(Fig. 2). Whether the primary reason for
this low PKC-
mRNA level in A/J lungs resulted from decreased mRNA
stability or reduced transcription of the PKC-
gene is not known.
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Inheritance pattern of PKC- content based on Mendelian crosses.
To determine the mode of pulmonary PKC inheritance, we crossed A/J and
B6 mice and examined progeny from F1, F2, and
both backcrosses. Lungs were harvested, and their PKC-
contents were determined (Table 2, Fig.
3). PKC concentration in the
(A×B6)F1 mice is intermediate to that in the A/J and B6
parents, analogous to the intermediate PKC content of lungs from
(A × CBy)F1 measured previously (22).
Most backcross progeny of the (A×B6)F1 × A backcross
have values resembling the A/J parent, whereas values of the
(A×B6)F1 × B6 backcross are distributed over the
range of both parental strains. This pattern is consistent with more than one gene being responsible for the strain difference in PKC-
content. In support of this, we compared the variances in the F1 and F2 generations to estimate the number of
genes contributing to the differential lung PKC content using the
formula n = (m1
m2)2/8(VF2
VF1) = (1.0
2.9)2/8(0.25
0.0009 = 1.8 genes, where m1 and m2
represent the mean values found in the parental strains,
VF2 and VF1 represent the variances of the F2
and F1 progeny, and the following assumptions were made: the genes are
unlinked, the genes are codominant, and the genes have equal
contributions to phenotype (33).
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A×B and B×A RI analysis of PKC- content and PKC activity.
PKC-
content and PKC activity in the A×B and B×A RI strains were
determined, and QTL analyses were performed; PKC activity and PKC-
content in these RI strains correlate well
(r2 = 0.74; P < 0.005;
Table 3). Lung PKC-
content and PKC
activity were each entered into Map Manager QT as quantitative traits
with the edited database of A×B and B×A prepared especially for
quantitative trait analysis (34). The major locus for
PKC-
content maps to chromosome 11, with the highest peak at
D11Mit333 (P < 0.0004; Fig.
4). This is the same location as the
PKC-
structural locus, although PKC-
has not been previously
mapped in these RI strains. The LRS for this map position is 12.5, which is approximately equal to a LOD score of 2.7. Permutation tests
give LRSs of 4.9, 15.3, and 24.8 as suggestive, significant, and highly
significant, respectively. A suggestive peak with a LRS of 6.2 at
D3Mit19 was found as well. The major locus for lung PKC
activity (Fig. 5), on the other hand,
maps to chromosome 3 at D3Mit19 with an LRS of 15.4 (P < 0.00008; LOD = 3.4). Permutation tests give
LRSs of 5.2, 14.2, and 25.0 as suggestive, significant, and highly significant, respectively. Additionally, there is a suggestive peak for
PKC activity with a LRS of 5.1 at D11Mit333.
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DISCUSSION |
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Of 22 inbred strains examined, only A/J mice had both low PKC-
content and PKC activity (Table 1). The inheritance pattern of PKC-
content elucidated with F1, F2, and backcrosses
with both A/J and B6 parents suggests that two genes are responsible for these strain variations (Table 2, Fig. 3). Although the correlation between PKC-
content and PKC activity among inbred strains was not
quite significant, content and activity among the A×B and B×A RI
strains did correlate with each other (r2 = 0.74; Table 3). The major determinant of PKC-
content mapped near
the PKC-
structural gene at 68 centimorgans on chromosome 11, with a
minor modifying gene that influenced PKC-
content at D3Mit19
or D3Mit94 (Fig. 4). The major determinant responsible for the low PKC activity in A/J mice mapped at 88 centimorgans on
chromosome 3, with a minor determinant at the PKC-
structural gene
locus Pkca (Fig. 5). Because the basis of the low PKC-
in A/J mice is a reduced level of PKC-
mRNA (Fig. 2), the chromosome 11 site responsible for this reduction could be the PKC-
promoter sequence, with the chromosome 3 site encoding a transcription factor
that binds differentially to the A/J versus B6 PKC-
promoters. Alternatively, A/J mice might have a polymorphism in the 3'-end of the
message that increases its interaction with ribonucleases, with the
chromosome 3 site encoding a protein that regulates mRNA stability by
binding to this 3' site.
The developmental expression of different PKC isozymes is strain,
isozyme, and tissue specific (38). An analogous situation to what we observed with PKC- in lung has been reported with hippocampal PKC-
. Within the hippocampus, the concentrations of six
PKC isozymes increased postnatally, whereas four others did not
(15). A strain dependence in the postnatal timing of PKC-
was observed wherein neonatal B6 and DBA mice have similar PKC-
content 10 days after birth, but enzyme content in B6 mice increased to a greater extent with age than in DBA mice
(30). This has been associated with the deficit in complex
learning and memory that DBA mice display relative to B6 mice. A
combination of deletion analysis and DNase footprinting was used to
examine the developmental expression of rat brain PKC-
(1). A 23-bp sequence within the 5' region was bound by
neonatal nuclear proteins to a greater extent than it was by nuclear
proteins from adult rat brains. Because PKC-
expression increased
with age, nuclear proteins that bound to this site correspondingly
decreased. Thus a cis-acting site repressed expression.
The basis of developmental regulation of the rate of protein synthesis among different cell types is not understood (3). Information has been obtained with transgenes consisting of a reporter construct attached to a host gene promoter. The degree to which transgene expression corresponds to expression of the endogenous gene with regard to cell specificity and age depends in part on the amount of ligated 5' sequence and whether any 3' or intronic sequence is also needed. A cis-acting site close to a structural gene plus another that may act cis or trans (i.e., reflecting the need for a diffusible product) is often required for normal ontogenic appearance. For example, if the 5' sequence ligated to a Clara cell-specific reporter was too short, the transgene was expressed prematurely during development (14).
The phenotype of an advanced tumor resembles its tissue of origin at an
earlier developmental stage more than it does adult tissue
(37). This is illustrated in human lung cancer where the
enzymatic content and isozyme distribution of dozens of enzymes were
more analogous to fetal lung tissue than to adult tissue (13). Mouse lung tumors have the low PKC- content that
is more characteristic of neonatal lung than of adult lung
(32). Genes that regulate the organ-specific appearance
during development of an enzyme involved in neoplastic conversion seem
reasonable candidates for susceptibility genes. One of the fourteen
genes that regulate susceptibility to lung tumorigenesis in mice is near the chromosome 11 site that regulates the postnatal appearance of
PKC-
(10). How might the low PKC-
content in A/J
mice contribute to their enhanced tumor susceptibility? The lungs of
A/J mice are unique in their exquisite sensitivity to induced and
spontaneous tumorigenesis (21), infection
(16), acute injury (31), and airway
hyperactivity (2). Tumor-promoting agents such as phorbol esters and butylated hydroxytoluene decrease PKC content in vitro (5), and pulmonary PKC-
content decreased 30 min after
butylated hydroxytoluene administration to mice (26).
Because high PKC-
levels are associated with a differentiated,
proliferatively quiescent phenotype in mouse lung epithelium
(6), low intracellular PKC-
concentrations might
predispose mice toward proliferative disorders such as neoplasia.
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ACKNOWLEDGEMENTS |
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We thank Cathy Auerbach and Kat Keil for excellent technical assistance.
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FOOTNOTES |
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This work was supported by a Parker B. Francis Fellowship (to L. D. Dwyer-Nield) and National Cancer Institute Grant CA-33497 (to A. M. Malkinson).
Address for reprint requests and other correspondence: A. M. Malkinson, Dept. of Pharmaceutical Sciences, School of Pharmacy, UCHSC, C-238, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: Al.Malkinson{at}UCHSC.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 10 November 1999; accepted in final form 29 March 2000.
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