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Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5; and the Departments of Obstetrics & Gynaecology and of Physiology, University of Toronto, Toronto, Ontario, Canada M5G IL4
Address all correspondence and requests for reprints to: Dr. Stephen J. Lye, Program in Development and Fetal Health, Samuel Lunenfeld Research Institute at Mount Sinai, 600 University Avenue, Suite 775, Toronto, Ontario, Canada. M5G 1X5. E-mail: Stephen_Lye{at}compuserve.com
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The rat osteosarcoma cell line UMR-106, which expresses the osteoblast phenotype, has been extensively used to study PTH actions (reviewed in Ref. 2). The PTH/PTH-related protein receptor signals through both the adenylate cyclase and phospholipase C (PLC) second messenger systems (5). Ligand binding to the G protein-coupled PTH receptor results in activation of adenylate cyclase, a rapid increase in cAMP and subsequent activation of protein kinase A (PKA) (6, 7). Activated PTH receptor also activates PLC, which subsequently produces diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (8). The second messengers DAG and IP3 mediate the activation of protein kinase C (PKC) (9), and the release of intracellular calcium (10). UMR-106 cells express Cx43 at a low basal level and respond to PTH treatment with an increase in Cx43 mRNA (4). Evidence suggests that PTH up-regulation of Cx43 mRNA in UMR-106 cells is transduced through the cAMP cascade because treatment with forskolin (an adenylate cyclase stimulator) or 8-bromo-cAMP (a cAMP analog) produces an effect similar to that of PTH (4). In contrast, the phorbol ester TPA (an activator of PKC) has no effect on Cx43 mRNA levels (11). However, the mechanisms by which PTH signaling increase Cx43 mRNA expression have not been determined.
The promoter region of the mouse Cx43 gene contains an activator protein-1 (AP-1) binding site, a target of both PKA and PKC signaling (12). This region shows a high degree of sequence homology to the promoter regions of both the rat and human Cx43 genes. We have previously shown that the AP-1 site can regulate basal expression of the promoter (13), and in the human myometrium this site confers responsiveness to TPA (14). In addition, we have identified positive and negative regulatory elements in the Cx43 promoter (13). Recently we have shown that Cx43 expression can be modulated by posttranscriptional mechanisms. The Cx43 3' untranslated region (UTR), which shows a high degree of sequence homology between mouse, rat, human, and bovine Cx43, is AU rich and contains four conserved AUUUA motifs that have been shown to confer instability to other transcripts (15). In SHM (Syrian hamster myometrium) cells, we have identified a 146-bp fragment between 2511 and 2656 in the Cx43 3'UTR that confers the highest basal expression (16). Therefore, both the promoter and 3'UTR of Cx43 contain potential regulatory regions that may be involved in the increase in Cx43 mRNA observed with PTH treatment. PTH signaling has been shown to cause both changes in the transcription rate and mRNA stability of target genes. In ROS 17/2.8 cells, PTH causes decreased transcription of the osteopontin gene (17), but an increase in the stability of osteocalcin mRNA (18). It has not been determined whether transcriptional and/or posttranscriptional mechanisms mediate the PTH-induced expression of Cx43.
In this study, we used in vitro and in vivo approaches to determine whether PTH-induced expression of Cx43 in the osteoblast cell line UMR-106 is mediated by transcriptional or posttranscriptional mechanisms. We report that sequences in both the promoter and the 3'UTR of the Cx43 gene are responsive to PTH. The PTH responsive sequences in the promoter were narrowed from the 5' end to -31 and from the 3' end to +1. In the case of the 3'UTR, the region between 2510 and 3132 showed the strongest response to PTH. In addition, we have determined that while the 3'UTR is responsive to the activation of both PKA and PKC signaling pathways, the promoter is only responsive to PKA activation. Therefore, both transcriptional and posttranscriptional mechanisms are important in mediating the increase in Cx43 expression induced by PTH treatment. Furthermore, these two mechanisms involve different signaling cascades.
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Materials and Methods |
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Cloning of deletion constructs
Various deletion constructs of the Cx43 promoter region were
ligated into a luciferase reporter gene vector. Previously, a 1.8-kb
segment of the Cx43 gene containing 1686 bp upstream and 165 bp
downstream of the transcription start site (-1686) was cloned from
mouse, and several deletion constructs were made from this promoter
segment (13). The constructs used in this study (-300,
-75, -54, -44, and -31) were identical with the large promoter
insert (-1686) except that the 5' region had been deleted respectively
at 300, 75, 54, 44, and 31 bp upstream of the transcription start site.
Similarly +97, +26, and +1 were generated by deletion of the 3' end of
the -300 segment respectively at 97, 26, and 1 bp downstream of the
transcription start site. These promoter segments were directionally
ligated into the luciferase reporter gene vector pGL2-basic(-)
[generated by removal of the SV40 3'UTR region (nt 1892–2743) from
the pGL2basic plasmid (Promega Corp., Madison, WI)], at
the HindIII and XhoI restriction sites.
Various regions of the rat Cx43 3'UTR [Cx43 complementary DNA (cDNA)
provided by Dr. E. C. Beyer, Washington University, St. Louis,
MO] were ligated into a luciferase reporter gene vector CMV-Luc. The
control vector for the 3'UTR experiments, CMV-Luc (originally termed
LUC(-)3'UT in 19) was generated by subcloning the cytomegalovirus
(CMV) promoter upstream of the luciferase gene in pGEM-luc
(Promega Corp.). The full-length rat Cx43 cDNA was
originally cloned from a rat uterine cDNA library (20).
The largest Cx43 3'UTR fragment containing 30 bp of the 3'-end of the
coding region and the full 3'UTR (nt 1311–3132) was ligated into
CMV-Luc at the StuI/SalI sites to generate the
Luc3132 construct. A series of fusion constructs were generated by PCR
using the full-length Cx43 cDNA clone as the template. The primers for
the constructs Luc1/3, Luc 2/3, Luc 3/3, Luc7/9, Luc8/9, and Luc9/9
were generated, so that the regions of the Cx43 3'UTR indicated in Fig. 7
were amplified and flanked by restriction sites for
XhoI and SalI. All constructs contained a 71-bp
fragment (nt 3062–3132) containing the polyadenylylation signal
(3107–3112). This fragment was generated by PCR and
inserted into the SalI/SfiI site downstream of
the subcloned fragments in all constructs. Finally the 3/3Luc construct
was generated by PCR using primers to amplify the region from
2510–3132 bp flanked by the NotI/BamHI
restriction sites, the resulting fragment was inserted upstream of the
CMV promoter in the CMV-Luc plasmid at the
NotI/BamHI sites.
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cells and purified for transfection using a QIAGEN Plasmid
Maxi Kit (QIAGEN, Mississauga, Ontario, Canada).
Transfection assay
The activity of the various Cx43 promoter constructs was
determined using a transfection assay. On day one, UMR-106 cells were
seeded at 200,000 cells/well in a 24-well plate, (Sarstedt, Newton, NC)
to ensure cells were in the log phase of growth when transfected. Cells
were transfected using 5 µl ExGen 500 cationic polymer, (MBI
Fermentas, Flamborough, Ontario, Canada) according to the product
protocol, in the presence of 0.5 µg of luciferase construct and 0.5
µg of pRSVßgal vector (ßGal, containing Escherichia
coli lacZ gene under the Rous sarcoma virus promoter). The cells
reached confluency 30 h after transfection.
Confluent cells were treated in triplicate with either sterile distilled water as a control or 5 nM PTH, the concentration previously shown to maximally stimulate Cx43 mRNA (4). For the time course experiment, control plates were harvested at time 0 and 5 h, and treated plates were harvested at 1, 2, 3, and 5 h of treatment. For the rest of the experiments, both control and PTH-treated plates were harvested at 5 h to obtain the greatest response. DMSO was used as a control for the 1 µM forskolin and 100 ng/ml TPA treatments, also performed in triplicate. At the appropriate harvest time, the media was removed, the cells were washed with PBS (Mg2+ and Ca2+ free) and harvested in 100 µl of reporter lysis buffer (Promega Corp.).
Luciferase activity of the cell lysates was determined using luciferin reagent (Promega Corp.) according to the manufacturer’s protocol. Luminescence was measured for 5 sec in a luminometer (Lumat LB9501, Berthold, Germany). Transfection efficiency was determined by assay for ßGal activity according to the method described in Hall et al. (21) and used to normalize luciferase activity. The activity of control vectors pSVLuc (pGL2-control, Promega Corp.) and CMV-Luc were used to normalize for differences between experiments for the promoter and 3'UTR analyses, respectively. Results shown are an average of at least four separate experiments, with error bars representing the SEM.
Transgenic mice
Microinjection was carried out using the -1686Luc construct
excised from the pGL2Basic vector. Gel purified DNA was microinjected
into male pronuclei of fertilized mouse eggs. Microinjections were
conducted on eggs harvested from superovulated ICR females mated with
ICR males. Transgenic founders were identified by luciferase assay as
described above and mated to ICR mice to obtain heterozygotes that were
bred to homozygosity by interbreeding. Eight-week-old heterozygous
pCx-1686Luc mice were treated with vehicle (sterile water) or 50 ng/g
body weight (BW) PTH by ip injection and killed by cervical dislocation
6 h after treatment. Expression of Cx43 and luciferase was
examined by immunolocalization. All animal experiments were approved by
the institutional animal care committee.
Immunohistochemistry
After 6 h of PTH treatment, mice were killed by cervical
dislocation, and tibia were removed and fixed in 10% formalin. Tissues
were then decalcified in 100 mg/ml [ethylenedinitrilo]-tetraacetic
acid disodium salt (EDTA; Mallinckrodt, Inc. Chemical,
Paris, KY), processed in ascending concentrations of ethanol,
cleared in xylene and embedded in bone wax (Paraplast X-Tra, Esbe Lab
Supplies, Toronto, Ontario, Canada). Sections were cut (5 µm),
de-waxed in xylene, rehydrated in a descending series of ethanol
solutions, and washed in PBS.
Cx43 expression was detected by immunofluorescence in the following manner. The tissue was permeabilized with 0.12% trypsin for 10 min at 37 C and washed in PBS. Nonspecific binding was blocked for 30 min using a solution of 5% goat serum (Vector Laboratories, Inc. Burlingame, CA) and 5% BSA (Sigma) in PBS. The tissue sections were then incubated overnight at 4 C in affinity-purified rabbit polyclonal anti-Cx43 antibody (raised against the c-terminus of Cx43, amino acids 360–382) diluted 1:150 in blocking solution. Sections were washed in PBS and then incubated for one hour in antirabbit fluoro-isothiocyanate (FITC; Zymed Laboratories, Inc., San Francisco, CA), diluted 1:60 in blocking solution. After final washes in PBS/0.02% Tween 20, sections were mounted with VectaShield (Vector Laboratories, Inc.) and viewed under UV light.
Luciferase expression was detected by immunohistochemistry in the following manner. After rehydration, endogenous peroxidase activity was inhibited by incubation in 1% hydrogen peroxide in methanol for 30 min, and slides were then washed in PBS. The tissue was permeabilized, and nonspecific binding was blocked as described above. Tissue sections were then incubated overnight at 4 C with rabbit polyclonal antiluciferase antibody (Promega Corp.) diluted 1:300 in blocking solution. Sections were washed in PBS and incubated for one hour in biotinylated goat antirabbit antibody (Vector Laboratories, Inc.), diluted 1:500 in blocking solution and then washed again in PBS. The tissue was then incubated for 2 h in the avidin-biotin peroxidase complex (ABC; Vector Laboratories, Inc.) and washed in PBS. Luciferase expression was detected using diaminobenzidine (DAB; Vector Laboratories, Inc.), and the tissue was counterstained with Harris Modified Hematoxylin (Fisher Diagnostics, Fair Lawn, NJ). Sections were then dehydrated, cleared in xylene and mounted with permount (Fisher Diagnostics).
Statistical analysis
Data from the time course experiments and forskolin/TPA
treatments were subjected to a one-way ANOVA followed by pairwise
multiple comparison procedures (Student’s-Newman-Keul’s method) to
determine differences between groups. Data from the deletion studies
were analyzed by two-way ANOVA followed by pairwise multiple comparison
procedures as described above. Where required the data were transformed
by the appropriate method to obtain a normal distribution. Statistical
analysis was carried out using SigmaStat version 1.01 (Jandel Corp.,
San Rafael, CA) with the level of significance for comparison set at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Cx43 promoter is responsive to PTH treatment in UMR-106
cells
To identify PTH responsive elements within the Cx43 promoter, a
full-length construct (-1686Luc) containing 1686 bp 5' of the Cx43
transcription start site and 162 bp of exon 1 was inserted into the
pGL2basic(-) luciferase vector. In addition, a series of 5' (-300,
-75, -54, -44, -31) and 3' (+97, +26, +1) deletion constructs were
prepared. As shown in Fig. 1, the
-1686Luc construct responds to PTH by 3 h with an increased
response 5 h after treatment. The cells treated with PTH for
5 h had a 4-fold higher luciferase activity than the control
cells. The 5 h control sample was not statistically different from
the 0 h control, indicating that the increase in luciferase
activity observed was due to PTH treatment (Fig. 1). Because the
greatest observed response was at 5 h, cells were harvested at
this time for all future experiments.
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, treatment of the -1686Luc transfected
cells with forskolin elicited a significant response, whereas TPA
treatment gave no response. Treatment with forskolin and TPA together
produced a response similar to that of forskolin alone, indicating that
TPA is not involved in stimulation of the Cx43 promoter even in the
presence of forskolin (Fig. 2). The response of the Cx43 promoter to
forskolin was only 2-fold higher than the control, whereas PTH elicited
a 3-fold response. This discrepancy could be due to the involvement of
other pathways in the signaling of the PTH receptor.
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). There is a dramatic drop in basal
luciferase activity with the deletion of the sequences between -75 and
-54, indicating the presence of a positive regulatory element. This
region contains the positive element we previously identified in SHM
cells (13). Deletion of the AP-1 site (-44Luc) and
further deletion to immediately before the TATA box (-31Luc) did not
affect the responsiveness to PTH (Fig. 3). To determine whether
sequences in exon 1 are responsible for PTH responsiveness, deletions
were also made in this region. As shown in Fig. 3, deletions up to 1 bp
3' of the transcription start site (+1Luc) did not disrupt the PTH
responsiveness. There appears to be a weak PTH responsive element in
the distal promoter because the ratio of PTH treated to control is 3.5
for the -1686 construct and between 1.8 and 2.6 for all other
constructs. However, the difference in these ratios was not
statistically significant. Although we have not been successful in
deleting the PTH responsive element in the Cx43 promoter, the
promoterless construct pGL2 basic(-), which is the parent vector for
all of our Cx43 promoter constructs, does not respond to PTH
(normalized luciferase activity: Control = 0.000116, 5
nM PTH = 0.000107). Therefore, the Cx43 promoter
sequences are responsible for the observed PTH responsiveness in these
transfection experiments. In addition, the positive control vector
pSVLuc containing an SV40 enhancer in the same parent plasmid did not
respond to PTH treatment. In summary, we have narrowed the sequences
required for PTH response from the 5' end to -31 and from the 3' end
to +1, and we have also ruled out that vector sequences are responsible
for this response. Comparison of the deletion analysis from the 5' and
3' ends resulted in an overlapping region from -31 to +1 that we
conclude is the important sequence for PTH response.
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). Luciferase expression was examined in
these tissue sections by immunohistochemistry using DAB detection.
Luciferase immunoreactivity was found to be higher in the tibial
sections of PTH treated mice, as demonstrated by an increase in
brown staining when compared with control mice (Fig. 4).
These results indicate the -1686 Cx43 promoter construct is capable of
conferring PTH responsiveness in vivo and elicits a response
similar to that of endogenous Cx43.
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). The cells
treated with PTH for 5 h had a 4-fold greater luciferase activity
compared with control cells (P < 0.05). The 5 h
control sample was not statistically different from the 0 h
control, indicating that the increase in luciferase activity was due to
PTH treatment (Fig. 5). Because the greatest observed response was at
5 h, cells were harvested at this time for all future
experiments.
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). The
response of the 3'UTR to TPA was similar to the PTH response, whereas
the response to forskolin was significantly lower. Forskolin and TPA
together did not respond at a greater extent than TPA alone, indicating
that forskolin and TPA do not have an additive effect (Fig. 6).
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). However the magnitude of the response
was 4.8-fold for Luc3/3 and only 2.6-fold for Luc1/3. When the ratio of
PTH treated: control was analyzed the response of Luc3/3 to PTH was
found to be significantly greater than that of both Luc1/3 and Luc3132.
The Luc3/3 construct was separated into three smaller regions, Luc7/9,
8/9, and Luc9/9. The Luc7/9 and Luc9/9 constructs showed a 2.7-fold
response to PTH, whereas the Luc8/9 construct showed no response (Fig. 7). Of all the 3'UTR constructs, Luc3/3 exhibited the greatest response
to PTH treatment. These results indicate that the 3/3 region contains
the major PTH responsive sequences and suggest that breaking this
region into smaller sections disrupts the PTH response.
The PTH responsive sequences within the Cx43 3'UTR do not function
as an enhancer
To determine whether the element in the 3'UTR required for PTH
response functions as an enhancer, the 3/3 region from 2510 to 3132 was
cloned 5' of the CMV promoter. The 3/3Luc construct had a higher basal
activity than the CMV-Luc negative control, which was not significantly
different from the basal activity of Luc3/3 (Fig. 8). Although the Luc3/3 construct
responds to PTH, the 3/3Luc construct showed no change with PTH
treatment (Fig. 8). The PTH-responsive element in the 3/3 region of the
3'UTR does not function in the 5' position and is therefore not likely
to work as an enhancer to increase the rate of transcription.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our studies examining the transcriptional regulation of Cx43 by sequences from 1686 bp upstream to 165 bp downstream of the transcription start site of the mouse Cx43 gene revealed that these sequences were responsive to PTH. Serial deletion of this region caused dramatic changes in basal luciferase activity as well as changes in the overall PTH-induced luciferase activity. However, deletions of this region did not significantly affect the 2- to 3-fold increase over basal observed with PTH treatment, indicating that the sequences responsive to PTH were not deleted. It has been suggested that the induction of c-fos is important in the regulation of genes responsive to PTH, because PTH treatment of UMR-106 cells results in a rapid induction of c-fos mRNA (reviewed in Ref. 2). The conserved AP-1 site in the Cx43 promoter may be a target for induction by c-fos, especially because this site appears to confer responsiveness to TPA in primary myometrial cells (14). Our data, however, do not support this assumption in UMR-106 cells because deletion of the AP-1 site did not significantly affect the 2- to 3-fold increase over basal in luciferase activity with PTH treatment. Our deletion studies have narrowed the region of PTH responsiveness in the Cx43 promoter to the region proximal to the TATA box. PTH responsiveness was present in constructs containing an overlapping region (-31 to +1 bp relative to the transcription start site), suggesting that this sequence contained the major PTH-response element. Recently there have been examples of transcription factors that interact directly with TATA binding protein or other components of the transcription machinery (reviewed in Ref. 23). Because we have narrowed the PTH responsive region to nucleotides surrounding the TATA box, we speculate that the transcription factors responsible for the PTH response may interact directly with proteins associated with the TATA box. Our studies suggest that other sequences including the AP-1 site (located within -54 to -44 bp) and an activator element (-75 to -54 bp, we previously identified in SHM cells) might also contribute to PTH-induced Cx43 expression. While not conferring specific responsiveness to PTH these elements dramatically alter basal promoter activity and thus the overall level of Cx43 expression following exposure to PTH.
Importantly, we have been able to confirm the physiological significance of these in vitro data in UMR-106 cells using mice carrying a transgene containing the full -1686 Cx43 promoter linked to a luciferase reporter gene. These mice displayed increased reporter gene and endogenous Cx43 expression in response to PTH in the region of bone underlying the growth plate. Gap junctional communication conferred by Cx43 has been shown to enhance physiologic responses to PTH (3). Taken together, our in vitro and in vivo data suggest that sequences proximal to the transcription start site are responsive to PTH and are likely important for providing coordinated responses to PTH within bone tissue.
Our analysis of the Cx43 3'UTR revealed the presence of sequences within the first one third (Luc1/3, 1311 to 2005 bp) and the final one third region (Luc 3/3, 2510 to 3132 bp) that respond to PTH treatment in UMR-106 cells. Although it is likely that multiple elements within these regions contribute to the overall response of the 3'UTR to PTH, we concentrated further analysis on the 3/3 region that exhibited significantly greater responsiveness to PTH than the full 3'UTR and the 1/3 region. Sequences within this region could cause the increase of the level of Cx43 mRNA through several mechanisms. They could act at the DNA level as an enhancer to increase the transcription rate of the Cx43 gene, or they could modulate Cx43 gene expression by increasing mRNA stability or by regulating the rate of translation initiation. Cloning of the 3/3 PTH responsive region 5' of the CMV promoter resulted in a loss of responsiveness to PTH, indicating that these sequences do not function as a classical enhancer. It is, however, possible that these sequences function to increase the rate of transcription but are not independent of position. Conversely, these sequences could function to regulate mRNA stability because other PTH responsive genes such as osteocalcin have been shown to be regulated by an increase in mRNA stability (18). In other studies, we have shown that the increased expression of Cx43 mRNA in SHM cells is correlated with increased mRNA stability mediated through this 3/3 region (16). However, experiments using actinomycin D in conjunction with PTH treatment showed no change in Cx43 mRNA stability in response to PTH (11). Thus, in UMR-106 cells, it would appear that the role of the 3'UTR of Cx43 in conferring increased responsiveness to PTH is most likely the result of an increased rate of mRNA translation. It has been suggested that an increase in translation rate in response to PTH allows for the increased expression of other genes, such as ornithine decarboxylase, in osteoblast cells (24).
Our studies thus indicate that sequences within the Cx43 promoter and 3'UTR can contribute to PTH-induced Cx43 expression through different mechanisms. This may not only result in greater overall PTH-responsiveness but may also provide the opportunity for regulation by different intracellular pathways. The PTH receptor signals through both PKA and PKC pathways (5). Previous studies in UMR-106 cells revealed that Cx43 mRNA expression is increased by cAMP analogues or activators of adenylate cyclase but not by activators of PKC (4, 11). Our demonstration that forskolin, but not TPA, is able to activate the Cx43 promoter is consistent with these studies examining endogenous Cx43 expression. In contrast to the effects on the Cx43 promoter, we have shown that the 3'UTR responds to both forskolin and TPA treatment. Although previous studies failed to show an effect of TPA on Cx43 expression in UMR-106 cells, these studies only measured changes in Cx43 mRNA (11). Our analysis of the 3'UTR suggests that PTH responsiveness of this region is likely through an increase in translation rate that would affect the level of Cx43 protein rather than mRNA. Our study suggests that the signaling pathways involved in the up-regulation of Cx43 in response to PTH treatment are complex and involve at least two distinct mechanisms. Studies in which cyclohexamide was shown to cause a partial but not complete attenuation of the response to PTH also suggested the existence of at least two mechanisms, one dependent on protein synthesis and one independent of protein synthesis (4). Although our studies have suggested mechanisms through which PTH can induce increased expression of Cx43, PTH can also increase gap junctional communication through cellular redistribution of the existing pool of Cx43 protein to the cell membrane (25). Taken together, these data suggest that PTH can increase gap junctional intercellular communication in osteoblast cells through actions at multiple levels including mRNA, protein and intracellular trafficking of the Cx43 protein. These multiple mechanisms are likely important in mediating both the immediate and sustained actions of PTH on Cx43 associated gap junctional communication and hence contribute to the overall biological actions of PTH on bone remodeling.
In summary, we have shown that sequences in both the Cx43 promoter and 3'UTR are involved in the response to PTH. We suggest that sequences in the Cx43 promoter region allow for an increase in transcription of the Cx43 gene in response to PTH. Conversely, the responsive sequences in the Cx43 3'UTR may act to increase the rate of translation, although further studies would be required to support this hypothesis.
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Acknowledgments |
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Footnotes |
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Received June 28, 2000.
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) are indicated.
Sections from control mice (A) showed very little punctate staining
compared with sections from PTH-treated mice (B), which displayed a
high level of punctate immunofluorescent staining for Cx43. Luciferase
staining of bone sections (100x) using DAB detection showed higher
luciferase immunoreactivity in sections from PTH treated mice (D)
compared with sections from control mice (C) as indicated by a higher
level of brown staining. A hematoxylin and eosin stained
section (E, 50x) shows the general region of the tibia underlying the
growth plate (GP) from which photomicrographs C and D were taken.
(P < 0.05). The 