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Cell Growth & Differentiation Vol. 11, 325-334, June 2000
© 2000 American Association for Cancer Research

Cell Cycle Control during Liver Development in the Rat: Evidence Indicating a Role for Cyclin D1 Posttranscriptional Regulation1

Michael M. Awad and Philip A. Gruppuso2

Brown University School of Medicine, Providence, Rhode Island 02912, and Department of Pediatrics, Division of Pediatric Endocrinology and Metabolism, Rhode Island Hospital, Providence, Rhode Island 02903


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Hepatocytes are capable of marked changes in proliferation in response to various physiological and pathophysiological stimuli. Although the changes in adult hepatocyte growth regulation that accompany reduction of liver mass, liver injury, and liver carcinogenesis have come under intense scrutiny, the regulation of hepatocyte growth during the latter stages of development is largely uncharacterized. We have examined hepatic cell cycle control in the developing rat. Analysis of term (fetal day 21) liver and cultured, term hepatocytes revealed G0-G1 growth-arrested cells relative to preterm (fetal day 19) liver and isolated hepatocytes. G1 cyclin-dependent kinase (CDK) activity was correlated with growth arrest at term in both in vivo and in vitro studies. The decline in CDK activity at term could not be attributed to a change in CDK protein content. Rather, the decline in CDK activity was associated with a concomitant decline in cyclin D1 protein content. However, cyclin D1 mRNA levels did not correlate with protein levels. Cyclin D1 mRNA was present at a higher level in adult livers, in which cyclin D1 protein was absent, than in fetal livers. We also examined the phosphorylation (activation) state of p38 mitogen-activated protein kinase, a potential hepatocyte-growth regulator and modulator of cyclin D1 content. p38 activity was inversely related to cyclin D1 content during liver development and regeneration. These data indicate that a posttranscriptional mechanism regulating cyclin D1 content is involved in the temporary hepatocyte growth arrest seen in the perinatal period and in the maintenance of adult hepatocytes in a quiescent state. We speculate that this posttranscriptional regulation may be downstream from the p38 mitogen-activated protein kinase pathway.


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
During the process of liver development, the transition from fetal to adult life is accompanied by a striking change in hepatocyte proliferation. In rodents, the last 3 days before birth is accompanied by a burst of hepatocyte proliferation that results in the tripling of liver mass and the replacement of the hematopoietic cell compartment by hepatocytes (1) . This high rate of hepatocyte proliferation is in marked contrast to the normally quiescent state seen in adult liver, in which only one in 20,000 hepatocytes undergoes mitosis under normal circumstances. Yet adult rat hepatocytes maintain the ability to proliferate after liver injury or reduction in liver mass (2) . Such changes in growth require precise and responsive cell cycle regulatory mechanisms.

Most of our current knowledge of these mechanisms in the liver is derived from experiments on adult rats in which liver mass is reduced by partial hepatectomy or injury, from models of hepatic carcinogenesis, or from studies on immortalized hepatocyte cell lines (3 , 4) . However, mechanisms regulating hepatocyte proliferation in these models may not be representative of those that are active during normal liver development. For example, available data indicate that growth factors such as hepatocyte growth factor, transforming growth factor {alpha}, and epidermal growth factor play a significant role in liver regeneration, probably acting through the MAP3 kinase signal transduction pathway (5) . In contrast, data from our laboratory indicate that fetal hepatocytes proliferate in the absence of exogenous growth factors, and that the low, constitutive level of MAP kinase activation that is seen during late gestation may be growth factor-independent (6) .

Studies using transgenic mice with homozygous gene deletions have often been used to derive information on the developmental role of various proteins. However such "knockout" experiments targeting cell cycle proteins have provided limited information. Deletions of most cyclins, most notably the G1 D- and E-type cyclins, result in few apparent developmental abnormalities, none involving the liver (7 , 8) . Exceptions include deletions of the G2 cyclins A2 or B1, which are lethal early in embryogenesis. Germ-line deletions of the CDKs have not been reported. Despite the importance of the CKIs in postnatal carcinogenesis, deletions of these genes are generally well tolerated during development, leading to only minor organomegaly of the pituitary, spleen, and thymus (9, 10, 11) .

Limitations with these models include early embryonic lethality prior to liver development and the lack of significant developmental effects, most likely attributable to redundancy among cell cycle proteins. To circumvent these issues, a transgenic mouse model was developed that specifically overexpressed the CKI p21Cip1 in postnatal hepatocytes (12) . Hepatocyte proliferation was inhibited dramatically in the postnatal period, which resulted in a reduction in the overall number of adult hepatocytes, aberrant tissue organization, decreased liver growth, decreased somatic body growth, and increased mortality. Significantly, the transgenic p21 protein was demonstrated to be associated with most, if not all, of the cyclin D1-CDK4 complexes in liver but not with other cyclin/CDK proteins, which emphasizes the importance of functional cyclin D1-CDK4 complexes as a part of normal liver development.

To elucidate the role of specific proteins and complexes during liver development, additional detailed studies of cell cycle protein expression and activity during the perinatal period are required. To this end, we have characterized the growth patterns of liver throughout development from late gestation through the adult period. Our earlier studies demonstrated an unusual ontogenic pattern of hepatocyte proliferation (13 , 14) . In vivo and correlative in vitro studies showed that the high rate of proliferation in preterm hepatocytes is followed by an abrupt decline at term with subsequent recovery of proliferation within 48 h of birth (13 , 14) . We have taken advantage of this observation by examining the content and activity of cell cycle constituents both during the period of temporary hepatocyte quiescence that occurs at term and during the transition from proliferating neonatal hepatocytes to quiescent adult hepatocytes. In doing so, we demonstrate G1 growth arrest in term fetal hepatocytes that is accompanied by a concomitant decline in cyclin D1-associated CDK activity. Furthermore, we have obtained evidence that this decrease is associated with inverse changes in cyclin D1 mRNA content, which indicates posttranscriptional regulation of cyclin D1 protein content in vivo. These results may pertain to mechanisms that maintain adult hepatocytes in a quiescent state. Our findings may, therefore, relate to pathophysiological and physiological perturbations that are capable of reactivating hepatocyte growth in the adult.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Term Rat Liver Is Arrested in G0-G1 Phase of the Cell Cycle.
To determine the phase of the cell cycle in which term hepatocytes are growth-arrested, flow cytometry of cells recovered from preterm, term, and adult rat liver sections was performed (Fig. 1A)Citation . Term liver obtained 6 h after birth demonstrated a high proportion of cells in G0-G1 phase, almost none in S-phase and few in G2-M relative to preterm liver cells. Adult liver cells also contained a large fraction of cells in G0-G1 and almost none in S-phase. There was a larger proportion of cells derived from adult liver that were identified as G2-M. This finding could be accounted for by the tetraploid nature of nonproliferating adult hepatocytes.



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  		Fig. 1. Flow cytometry analysis of the cell cycle status of developing hepatocytes. Flow cytometry was performed on cells isolated from fixed liver (A), and on freshly isolated hepatocytes (B). Graphs represent the flow cytometry results as DNA content versus number of cells [left-most gray peak, G0-G1; central black peak (when present), S phase; right-most gray peak, G2-M]. Percentages below each graph, the proportion of cells in each phase of the cell cycle. These results are representative of triplicate experiments.

 
To confirm the cell cycle status of term hepatocytes, flow cytometry was performed on freshly isolated preterm and term hepatocytes (Fig. 1B)Citation . One-quarter of preterm hepatocytes were found to be traversing S or G2-M phase. In contrast, fewer than 10% of term hepatocytes were found to be in S phase or G2-M. Nonetheless, both sets of analyses were interpreted as showing G0-G1 hepatocyte growth arrest at term. Our previous findings comparing bromodeoxyuridine incorporation in cultured preterm versus term hepatocytes (14) showed that DNA synthesis was asynchronous in preterm hepatocyte cultures. In contrast, term hepatocytes showed minimal DNA synthesis on the first day in culture and entered S-phase synchronously on the second day in culture. These prior results, which indicated growth arrest that was relieved when cells were isolated, are consistent with the present flow cytometry findings that indicate G0-G1 growth arrest at term.

G0-G1 Growth Arrest at Term Is Associated with a Decrease in G1 CDK Activity.
Passage through the G1 phase of the cell cycle is dependent on activation of the CDKs CDK4 and/or CDK6 and the resulting phosphorylation of the retinoblastoma gene product pRb (15) . Therefore, we measured CDK4 and CDK6 activity in preterm and term whole liver homogenates and cultured hepatocyte lysates. CDK4 and CDK6 activities were determined using an in vitro IP kinase assay with GST-pRb as the kinase substrate. CDK4 activity decreased dramatically in term liver when compared with preterm liver (Fig. 2A)Citation . Activity in adult liver was negligible. CDK6 activity paralleled the pattern for CDK4 (data not shown). However, signal intensities were very low for reasons that are not clear.



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  		Fig. 2. CDK4 activity in vivo and in vitro. IP kinase assays were performed on whole-liver homogenates (A) and lysates of fetal hepatocytes cultured for the indicated times [B, E19 (filled bars) and E21 (unfilled bars)]. Activity was measured as incorporation of 32P from [{gamma}-32P]ATP into GST-pRb fusion protein. Representative autoradiograms are shown (lower panels) with corresponding densitometry (graphs). Error bars, SE. Results were confirmed in three additional experiments. C1, minus antibody control; C2, minus sample control; C3, minus substrate control.

 
Lysates of preterm and term hepatocytes cultured for up to 78 h were also assayed for CDK4 activity (Fig. 2B)Citation . In both of them, activity within 6 h of isolation was low. Activity in preterm hepatocytes began to rise between 6 and 12 h in culture and reached peak CDK4 activity at 30–36 h in culture. In contrast, term hepatocytes did not demonstrate increased CDK4 activity until 54 h in culture, peaking near the end of the experiment.

Activity of CDK4 and CDK6 is dependent on association with G1 cyclins, most notably cyclin D1 (16) . To assess the involvement of cyclin D1 in active G1 CDK complexes, we immunoprecipitated cyclin D1 from preterm, term, and adult whole liver homogenates and used GST-pRb phosphorylation as a measure of cyclin D1-associated kinase activity (Fig. 3)Citation . Kinase activity was high in preterm liver and nearly undetectable in term and adult liver.



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  		Fig. 3. Cyclin D1-associated kinase activity. IP kinase assays for GST-pRb phosphorylation were performed on E19, E21, and adult whole liver homogenates immunoprecipitated with cyclin D1 antibodies. Densitometry is shown as the mean plus SE. Lower panel, a representative autoradiogram. Control conditions are those described for Fig. 2Citation . Similar results were obtained in two additional experiments.

 
Hepatocyte Quiescence at Term Is Associated with Decreased Nuclear Cyclin D1 and Cyclin E Protein Content.
The activity of G1 CDKs requires the presence of nuclear complexes of cyclin D1 with CDK4 and CDK6 (16) . To determine whether the marked decrease in perinatal CDK activity might be attributable to a decrease in content of one or of more than one component, we measured both cyclin D1 and CDK4/CDK6 nuclear protein content and complex formation. Nuclear extracts were prepared from preterm, term, and adult livers. Nuclear CDK4 content was determined by direct Western blot analysis. Nuclear CDK4 protein levels showed a modest decline throughout the developmental period studied (Fig. 4A)Citation . CDK6 protein levels could not be assessed by direct immunoblotting, presumably attributable to either low sensitivity of the CDK6 antibodies or low CDK6 content. Therefore, these analyses were performed using IP followed by Western blotting. CDK6 nuclear protein content declined as gestation proceeded (Fig. 4B)Citation . Levels in adult liver were similar to those seen in growth-arrested term liver. In both cases, CDK content did not decline to a degree that correlated with the changes in kinase activity.



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  		Fig. 4. CDK protein content in liver nuclear extracts. In A, nuclear extracts prepared from livers of various developmental ages were analyzed by Western blot for CDK4. The result shown was replicated in a second, independent experiment. In B, parallel analyses were done for CDK6 using IP followed by Western immunoblot. Upper panel, densitometric analysis of the autoradiogram shown in the lower panel. Error bars, SE; C1, no antibody control. Similar results were obtained in two replicate experiments.

 
Coimmunoprecipitation assays of CDK6 with cyclin D1 from perinatal and adult whole liver homogenates were performed to assess the level of G1 cyclin-CDK complexes. Significant levels of cyclin D1-associated CDK6 were detected in preterm liver. In contrast, cyclin D1-immunoprecipitable CDK6 was nearly undetectable in term fetal and adult livers (Fig. 5)Citation . In parallel experiments, cyclin D1-associated CDK4 could not be detected in any samples. Again, our data did not provide an explanation for this, although it is possible that the epitope recognized by the cyclin D1 antibody is made inaccessible by binding to CDK4.



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  		Fig. 5. Cyclin-CDK complexes in preterm, term, and adult liver. Whole-liver homogenates were immunoprecipitated with anticyclin D1 and then analyzed by Western immunoblotting for CDK6. The graph shows densitometric analysis of the resulting autoradiogram (inset). Error bars, SE.

 
The decrease in cyclin D1-immunoprecipitable CDK6 protein despite a modest decline in nuclear CDK6 levels suggested a block in cyclin-CDK complex formation. This is most commonly attributable to the presence of a CKI or the absence of cyclin D1 available for complex formation (17) . Cyclin D1 protein levels were analyzed by Western blot of unfractionated liver homogenates and nuclear extracts. Cyclin D1 levels in whole liver homogenates were highest on E17, markedly reduced in the perinatal period (E21, P1), and slightly increased in the immediate postnatal period (Fig. 6A)Citation . Cyclin D1 was undetectable in adult liver homogenates. A similar pattern was observed for cyclin D1 protein levels from nuclear extracts (Fig. 6B)Citation . As for whole liver homogenates, no cyclin D1 of Mr 34,000 could be detected in adult nuclear extracts. However, several higher molecular weight immunoreactive bands ranging from Mr 36,000 to 46,000 were consistently observed in adult nuclear extracts. Attempts to recover these forms by IP and to detect them in immunoblots have been unsuccessful. Thus, their relationship to cyclin D1 is uncertain.



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  		Fig. 6. Ontogeny of hepatic G1-cyclin protein content. Densitometry of Western immunoblotting analysis of cyclin D1 from whole-liver homogenates (A), cyclin D1 from nuclear extracts (B), and cyclin E from nuclear extracts (C) is shown with accompanying representative autoradiogram (insets). P.25, 6 h after birth; P.75, 18 h after birth. Results for all of the experiments were replicated three times.

 
Cyclin E-CDK2 complexes are required for cell cycle progression through late G1 and for the G1-S transition (18) . Cyclin E expression is dependent on and succeeds the expression of cyclin D1 and subsequent activation of CDK4 and/or CDK6 early in G1 phase (19) . As an additional indicator of cyclin D1 down-regulation, cyclin E levels were assessed by Western blot analysis of nuclear extracts. Cyclin E protein levels were maximal in preterm liver, declined markedly in term liver, and were negligible in adult liver (Fig. 6C)Citation .

Cyclin D1 Down-Regulation Occurs in Term Hepatocytes in Vivo.
In the immediate prenatal period, ~50% of the cells in liver are of hematopoietic origin (1) . To determine which cell types contribute to the perinatal decrease in liver cyclin D1 protein content, cyclin D1 immunohistochemistry of preterm and term liver was performed. In preterm liver, >85% of liver cells demonstrated intense staining for cyclin D1 (Fig. 7A)Citation . In contrast, <15% of cells were cyclin D1-positive in term liver, and staining was significantly less intense in positive cells (Fig. 7B)Citation . Individual hepatocytes in preterm and term liver were identified by morphological phenotype and counted for cyclin D1 staining. Results showed that the decrease in cyclin D1 staining in hepatocytes from preterm to term paralleled that of all of the cells (85 versus 15%).



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  		Fig. 7. Cyclin D1 indirect fluorescent immunohistochemistry of E19 (A) and E21 (B) liver sections (x200).

 
Discordance between Cyclin D1 mRNA Content and Cyclin D1 Protein Levels during Liver Development.
Cyclin expression has been shown to be regulated at transcriptional, posttranscriptional, and posttranslational levels (20, 21, 22, 23) . We, therefore, examined whether mRNA levels parallel the decrease in liver cyclin D1 and cyclin E protein levels as fetuses approach term. Total RNA from preterm, term, and adult livers were analyzed by Northern blot (Fig. 8A)Citation . Surprisingly, the 4.4-kb RNA detected was most highly abundant in the three adult liver RNA preparations that we analyzed. The same RNA species was present in very low levels in the RNA preparations from preterm and term fetal liver. This pattern of expression could not be accounted for by RNA loading. Given the very low cyclin D1 mRNA level in fetal liver, we proceeded to confirm these results by relative quantitative RT-PCR using the primer-dropping method (Fig. 8A)Citation . This method has the advantage of higher sensitivity and specificity, assured by confirming the sequence of the cyclin D1 PCR product. Results showed that cyclin D1 mRNA levels were similar in preterm and term fetal liver. Again, the increase in adult liver was found in triplicate analyses. In addition, we analyzed RNA preparations from a more complete panel of liver samples (Fig. 8C)Citation . Results showed that a marked increase in cyclin D1 mRNA content occurred between the end of gestation and the end of the first postnatal week, by which time adult cyclin D1 mRNA levels had been attained. To further confirm the validity of the analyses, we also examined cyclin D1 expression after partial hepatectomy. As expected, cyclin D1 mRNA was markedly induced 24 h after partial hepatectomy compared with sham operation. In contrast to cyclin D1, cyclin E mRNA paralleled cyclin E protein levels, being highest in preterm liver, lower in term liver, and nearly undetectable in adult liver (Fig. 8D)Citation .



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  		Fig. 8. G1-cyclin mRNA levels. Total RNA isolated from rat liver was analyzed for cyclin expression levels. Northern analysis was performed for cyclin D1 using subcloned PCR product as a probe (A, upper panel). rRNA levels on the ethidium bromide-stained gel were used as a loading control (A, lower panel). To confirm these results, multiplex semiquantitative RT-PCR was performed to assess cyclin D1 mRNA content at the three developmental time points analyzed by Northern blot (B). An additional experiment (C) was performed in which cyclin D1 mRNA content in single samples from an E19 fetal rat; from rat pups on postnatal (P) days 1, 4, 7, 14, and 28; and from a normal adult rat (Ad). This experiment also included analysis of RNA preparations from two adult rats that underwent partial hepatectomy (PH) or sham operation (S) 24 h before sacrifice. For these last three samples, the number of PCR cycles was reduced to keep amplification in the linear range. D, an experiment in which a parallel analysis was carried out for cyclin E.

 
Temporal Association between p38 MAP Kinase Activity and Cyclin D1 Protein during Liver Development and Regeneration.
On the basis of available data (see "Discussion"), we hypothesized a potential role for the p38 MAP kinase pathway in the regulation of cyclin D1. Although our in vivo liver development model is not conducive to studies that could place cyclin D1 downstream from p38, we did take advantage of our prior characterization of hepatocyte proliferation and cell cycle events during the perinatal period. The activity state of p38 was determined indirectly by Western immunoblotting with antibodies that recognize the active, phosphorylated form of the enzyme. Results (Fig. 9)Citation showed an increase in phospho-p38 at term. This was not associated with a change in total p38 content. At 6 and 24 h after partial hepatectomy, p38 was under-phosphorylated compared with its state in livers from sham-operated animals (Fig. 9)Citation . By 48 h after partial hepatectomy, p38 was phosphorylated to a similarly high degree in both regenerating liver and liver from sham-operated animals. These results demonstrate that p38 activation, as determined by its phosphorylation state, is inversely related to cyclin D1 content during normal development. The finding that it is inactivated after partial hepatectomy supports the hypothesis that it functions as a tonic growth inhibitor in adult liver.



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  		Fig. 9. Regulation of p38 MAP kinase during liver development and liver regeneration. Liver homogenates were analyzed by Western immunoblotting using primary antibody specific for phosphorylated (active) p38 (P-p38) or total p38. Samples were obtained from fetal rats on E17, E19, or E21. Postnatal samples were obtained 1, 4, 7, and 28 days after birth (P1, P4, P7, P28). Additional samples were obtained at various times after partial hepatectomy (PH) or sham operation (S).

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Extraordinary progress has been made over the last decade in understanding the means by which cell replication is controlled. With regard to liver biology, many studies have focused on the model of liver regeneration after partial hepatectomy (24, 25, 26) . However, relatively few studies have focused on mechanisms of cell cycle control under normal physiological conditions associated with marked changes in rates of cell proliferation, such as those seen during the perinatal and neonatal-to-adult transitions in the rat. Hepatocyte proliferation proceeds rapidly during late gestation and the immediate neonatal period in the rat, with a period of temporary quiescence occurring at term (12, 13, 14) . A transition to a quiescent adult hepatocyte phenotype takes place by the end of the first postnatal week. The present studies were undertaken with the premise that hepatocyte cell cycle regulation during normal development involves mechanisms that are relevant to other aspects of liver biology.

On the basis of the results from flow cytometry analyses, we focused on the G1 phase of the cell cycle as the control point for perinatal hepatocyte growth arrest. The difference in flow cytometry results when comparing the whole liver, and cell culture experiments may be accounted for by the presence of a significant hematopoietic component in the fetal whole-liver samples (1 , 27) , which is nearly absent in primary fetal hepatocyte cultures (13) . Alternatively, hepatocyte isolation may have affected the cell cycle status of isolated hepatocytes. Of note, we found a significant proportion of cells in adult liver with 4 N DNA content. This is attributable to the increased ploidy (binucleate diploid and mononulceate tetraploid) typically seen in postnatal hepatocytes (27) . These cells were indistinguishable from G2-M phase cells in our analyses.

Our data indicate that, in particular, cyclin D1 can be assigned a key G1-phase regulatory role in perinatal hepatocyte growth arrest. IP kinase assays showed growth-associated changes in the activity of the early G1-phase cyclin D-dependent kinases, CDK4 and CDK6, without which G1-phase cannot progress (28) . Our in vivo analyses were supported by in vitro results showing that preterm (E19) hepatocytes possess significant CDK4 activity during the first day in culture, whereas CDK4 activation was delayed by 36–48 h in term hepatocytes. This pattern is consistent with our earlier findings (14) , which suggest that term hepatocytes are under a growth-inhibitory influence in vivo and, once relieved from this influence, spontaneously enter the cell cycle in culture without exposure to serum or growth factors.

In term livers, pRb kinase activity in cyclin D1-immunoprecipitated complexes was similarly diminished. Potential mechanisms for the inhibition of CDK activity include the absence of G1 cyclins and/or CDKs, prevention of complex formation by the action of CKIs, or inhibition of the kinase activity of formed cyclin D1/CDK complexes by CKIs (29) . CDK4 and CDK6 proteins could be detected in nuclear extracts at levels that would not be limiting for the formation of cyclin-CDK complexes. This is consistent with the manner of posttranslational regulation by which CDKs are regulated in other systems. It should be noted, however, that the presence of G1-phase CDKs in adult liver nuclear preparations has not been described previously. This observation was unexpected given the view that quiescent, mature hepatocytes are generally considered to be arrested in G0. Persistent hepatic CDK expression in the adult may relate to the observation that hepatocytes have considerable potential for rapid reactivation of growth after growth stimuli or liver injury (5 , 25 , 30) .

In contrast to G1 CDK levels, cyclin D1 protein levels varied considerably and in parallel with hepatocyte proliferation, being lowest in term and adult liver preparations. Similarly, cyclin E levels correlated with hepatocyte growth arrest. This finding is consistent with the role of cyclin E as a late G1-S cyclin that follows and is dependent on early G1 cyclin D-associated complex activity (19 , 31 , 32) . Whereas there is a significant decline in the proportion of hematopoietic cells from preterm to term liver (1 , 27) , this did not account for the decrease in hepatic cyclin D1 content based on the results of immunohistochemistry.

Published studies (33 , 34) have demonstrated the involvement of the CKIs p21 and p27 in regulation of CDK activity during liver regeneration. It is likely that these growth modulators play a role in normal liver development. However, their ability to regulate CDK activity presumes the presence of the requisite cyclin required for any particular CDK. We have performed preliminary studies on the expression of CKIs during the perinatal period.4 Multiple CKIs show changes in their expression during the 48 h before, and the week after, parturition. Whereas these findings may indicate a role for CKI expression in the control of hepatocyte proliferation during development, it is unlikely that they supplant the role of cyclin D1 down-regulation at times of growth arrest, because cyclin D1 content would be limiting at these times. This is in contrast to the acute growth stimulation seen during liver regeneration after partial hepatectomy. Under these circumstances, CKIs of the Cip/Kip family might be required for cyclin/CDK complex formation (35) .

Analysis of steady-state mRNA levels was used to determine whether the regulation of G1 cyclins was transcriptional or posttranscriptional. Whereas cyclin E mRNA levels paralleled the profile for cyclin E protein content, cyclin D1 mRNA levels did not decrease in maturing liver. In fact, cyclin D1 message levels were elevated in adult liver relative to earlier developmental time points. As was the case for the high adult liver CDK4 and CDK6 content, this result was unexpected given the quiescent state of adult liver. Again, this indicates that the resting state of quiescent adult rat hepatocytes differs markedly from the G0 state seen in other well-characterized cells, such as fibroblast cell lines, in which expression of G1 cell cycle proteins is absent or markedly diminished (36, 37, 38, 39, 40, 41, 42) .

Posttranscriptional regulation of cyclin D1 has been described previously in NIH3T3 cells and in immortalized bronchial epithelial cell lines (20, 21, 22) . It has been suggested that this is mediated in NIH3T3 cells at the translational level by cyclin D1 message interaction with the eukaryotic initiation factor, eIF4E. Cyclin D1 posttranscriptional regulation in an in vivo model, liver regeneration after partial hepatectomy in the rat, has been proposed (26) but not demonstrated.

The mitotic cyclins A and B have been well characterized with regard to their modification and subsequent degradation by ubiquitin-mediated proteolysis on the completion of the cell cycle (43, 44, 45) . More recently, the ubiquitin-proteosome pathway has been assigned a role in the control of cyclin D and cyclin E content (46, 47, 48) . Whereas the Mr 34,000 cyclin D1 bands detected by Western immunoblotting disappeared over the course of postnatal development, several cyclin D1-immunoreactive bands ranging from Mr 36,000 to 46,000 were consistently observed in adult liver nuclear extracts. It is possible that these higher molecular weight bands represent a modified form of cyclin D1. It is possible that higher molecular weight cyclin D1 immunoreactive proteins may be an indication that the observed posttranscriptional regulation of cyclin D1 content involves cyclin D1 ubiquitination during liver development and in the maintenance of the quiescent state in adult hepatocytes.

Our results do not define a mechanism for the posttranscriptional regulation of cyclin D1 during development. However, our data do suggest that cyclin D1 posttranscriptional regulation has a key growth-regulating role in liver development. This is consistent with the findings of Albrecht and Hansen (49) , who showed that overexpression of cyclin D1 in primary cultures of adult rat hepatocytes was sufficient to promote progression through the G1 restriction point. With regard to the upstream mechanisms controlling cyclin D1 content, we were led to examine a possible role for the p38 MAP kinase pathway based on studies that have defined this signaling kinase as mediating growth inhibition via an effect on cyclin D1 (50) . In addition, we were influenced by data showing that p38 can mediate posttranscriptional regulatory events (51) . Our preliminary data examining the regulation of p38 activity demonstrate a pattern that is inversely related to cyclin D1 abundance. On the basis of this, we have performed subsequent studies that indicate that p38 activation in cultured fetal hepatocytes can down-regulate cyclin D1 content.5 The direct demonstration that this is mediated by posttranscriptional events will require further investigation. Nonetheless, the present studies strongly support the physiological relevance of cyclin D1 to physiological hepatocyte growth regulation, including a contribution of posttranscriptional control.


   Materials and Methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Animals.
Pregnant Sprague Dawley rats (Charles River Breeding Laboratory, Wilmington, MA) of known gestational age (day of conception designated as E0, preterm as E19, and term as E21) were used for all of the studies. Given the importance of precisely determining timing at the end of gestation, term animals were identified by frequent observation for onset of parturition. Partial (two-thirds) hepatectomy was carried out on adult male rats (125–150 g) under methoxyfluorane anesthesia. Sham-operated animals underwent liver exteriorization without excision.

Hepatocyte Isolation and Primary Culture.
Fetal and newborn rat hepatocytes were isolated by collagenase digestion as described previously (13) . Immunocytochemical analyses (13) have demonstrated that these preparations consist of ~90% hepatocytes, with the remaining cell population consisting of a mixture of nonparenchymal cell types. This level of hepatocyte predominance persists for up to 78 h in culture under the defined mitogen-free conditions used for all of the experiments.

Hepatocytes were cultured on Falcon Primaria plates (Becton Dickinson, Franklin Lakes, NJ) at a density of 2 x 106 cells per 100-mm plate. Cells became attached within 2 h in MEM containing 5% fetal bovine serum and supplemented with nutrients and cofactors, as described previously (13) . After cell attachment, all of the studies were done in defined (serum-free), supplemented MEM.

Preparation of Hepatocyte Lysates, Whole Liver Homogenates, and Nuclear Extracts.
Preparation of hepatocyte lysates was carried out as described previously (28) . Briefly, cultured cells were rinsed twice with 10 ml of cold PBS and then scraped into 2 ml of IP buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, and 0.1% Tween 20] containing 10% glycerol, 144 µM AEBSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 mM ß-glycerophosphate, 1 mM NaF, and 0.1 mM sodium orthovanadate. Lysates were then sonicated at 4°C (full microtip power twice for 10 s each time; Ultrasonic Homogenizer 4710 Series, Cole-Parmer, Chicago, IL) and clarified by centrifugation at 10,000 x g for 5 min at 4°C.

For preparation of whole liver homogenates, the pooled livers from one litter were combined in 1 ml per 100 mg tissue cold IP homogenization buffer without Tween 20. Tissue was homogenized for 10 strokes at 700 rpm using glass-teflon homogenization vessels. Tween 20 was then added to a final concentration of 0.1%. Homogenates were clarified by centrifugation at 10,000 x g for 10 min at 4°C, frozen immediately on dry ice, and stored at -70°C.

For preparation of whole-liver nuclear extracts, 0.5–1.0 g of pooled liver was homogenized in 5 ml of buffer A1 [15 mM HEPES (pH 7.5), 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 14 mM 2-mercaptoethanol, 10 mM NaF, 1 mM sodium orthovanadate, 144 µM AEBSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin] with 5 strokes at 800 rpm in glass-teflon homogenization vessels. Homogenates were allowed to settle on ice for 5 min, and then the top 4 ml was centrifuged at 700 x g for 5 min at 4°C. The resulting supernatant was resuspended in 5 ml buffer A2 (A1 with 250 µl of NP40 per 50 ml) and centrifuged over 5 ml of buffer B [15 mM HEPES (pH 7.5), 30% sucrose, 60 mM KCl, 15 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, and 14 mM 2-mercaptoethanol] for 5 min at 1500 x g at 4°C. Pelleted nuclei were resuspended in IP buffer. Lysates were clarified by centrifugation at 10,000 x g for 15 min at 4°C. Samples were frozen on dry ice and stored at -70°C.

Flow Cytometry.
For preparation of cells from fixed livers, tissue was suspended in IP buffer diluted with an equal volume of pepsin solution [140 mM NaCl and 5 mg of pepsin per ml of solution (pH 1.5)], incubated at 37°C for 30 min with high-speed vortexing every 5 min, and incubated in 2.5 volumes trypsin solution {120 µg of trypsin per ml citrate buffer [14 mM sodium citrate, 2 mM Tris, 0.4% (v/v) NP40] and 10 mM spermine (pH 7.6)} for 10 min at 20°C. Trypsin digestion was stopped by incubation with 0.6 volumes trypsin inhibitor solution (0.1 g of trypsin inhibitor, 0.02 g of RNase A per 50 ml citrate buffer) for 10 min at 20°C. Cells were stained with 0.4 volumes propidium iodide solution containing 0.083 g of propidium iodide and 0.23 g of spermine per 50 ml of citrate buffer) for 15 min in the dark at 20°C and then analyzed.

For preparation of cultured hepatocyte suspensions, cells were scraped from 100-mm plates into 2 ml of cold PBS, washed with 2 x 10 ml of cold PBS, and resuspended in PBS. Cells (2.0 x 106) were pelleted and resuspended in 1.0 ml of propidium iodide solution [7.5 µM propidium iodide, 0.1% (v/v) NP40, and 0.1% (w/v) sodium citrate] for 15 min in the dark at 20°C and then analyzed.

All of the flow cytometric analyses were performed on a FACScan Flow Cytometry System (Becton Dickinson, Franklin Lakes, NJ).

IP and CDK Assays.
IP and kinase assays were performed as described by Matsushime et al. (28) with minor modifications. Briefly, 4 mg of liver homogenate protein or 100 µg of hepatocyte lysate protein were immunoprecipitated for 2 h at 4°C with protein A-Sepharose beads cross-linked to saturating amounts of the indicated antibodies (52) . For kinase assays, immunoprecipitated proteins on beads were washed four times with 1 ml of IP buffer and twice with 50 mM HEPES (pH 7.5) containing 1 mM DTT. The beads were suspended in 30 µl of kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM DTT] containing substrate [1 µg of soluble GST-pRb fusion protein (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)], 2.5 mM EGTA, 10 mM ß-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF, 20 µM ATP and 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; NEN DuPont, Boston, MA). Results were validated by the use of three control conditions: omission of antibody in the IP reaction (no antibody control), omission of sample, and omission of kinase substrate. After incubation for 30 min at 30°C with occasional mixing, the samples were boiled in PAGE sample buffer containing SDS and were separated by PAGE. Phosphorylated proteins were visualized by autoradiography of the dried gels.

PAGE and Western Blot Analyses.
Liver proteins were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). CDKs and cyclins were detected using antibodies obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). In particular, the cyclin D1 antibody does not cross-react with cyclins D2 or D3. For Western immunoblot detection of phosphorylated (active) p38 MAP kinase and total p38 MAP kinase, primary antibodies were obtained from New England Biolabs (Beverly, MA). For all of the Western blots, we used peroxidase-conjugated secondary antibody followed by chemiluminescent detection with the ECL Plus detection system (Amersham, Inc., Piscataway, NJ).

Immunohistochemistry.
Formalin-fixed liver sections (6-µm) were covered with 10 mM sodium citrate buffer (pH 6.0) and were heat-treated at 95°C twice for 5 min. Sections were treated with avidin/biotin blocking solutions (Vector Laboratories, Burlingame, CA), and then with 5% normal horse serum (Life Technologies, Inc., Gaithersburg, MD) in PBS (15 min each at room temperature). Sections were then incubated with 20 µg/ml cyclin D1 primary antibody (sc-8396; Santa Cruz Biotechnology, Inc.) in PBS for 30 min at room temperature followed by incubation with horse antimouse secondary antibody (1:500 dilution; Vector Laboratories). Signal was detected after incubation with fluorescein-streptavidin conjugate (Vector Laboratories).

Relative Quantitative RT-PCR.
Total RNA was isolated from frozen liver samples by homogenization in guanidium isothiocyanate followed by cesium chloride density centrifugation (53) . cDNA was generated using 3 µg of total RNA in the Superscript Preamplification System for First-Strand cDNA Synthesis kit (Life Technologies, Inc.). Primer-dropping PCR was performed as described previously (54) . Primer sequences used for detection of rat cyclin D1 transcripts were 5'-GCGTACCCTGACACCAATCT-3' for the sense primer and 5'-GCTCCAGAGACAAGAAACGG-3' for the antisense primer, resulting in a predicted PCR product size of 232 bp. These primers do not recognize other D-type cyclins. Primers used to detect rat cyclin E transcripts were 5'-ATGTCCAAGTGGCCTACGTC-3' for the sense primer and 5'-CTTTCTTTGCTTGGGCTTTG-3' for the antisense primer, resulting in a predicted PCR product size of 375 bp. PCR products were sequenced to confirm identity (Yale University Keck Biotechnology DNA Sequencing Laboratories, New Haven, CT). Control rat ß-actin primers were obtained from Clontech, Inc. (Palo Alto, CA). Optimal PCR cycle numbers, required for exponential amplification for each primer set, were determined by preliminary range-finding experiments. Total amplification in each multiplex reaction was kept below saturation levels to permit the products to remain within the exponential range of the amplification curve and, thereby, provide semiquantitative data. Gels were illuminated with UV light, photographed, and analyzed by digital image analysis. All of the PCR experiments were performed in triplicate to verify results.

Northern Blot Analysis.
Total RNA was isolated as described above. Total RNA (20 µg) was separated on a 1% formaldehyde-agarose gel, followed by transfer to nylon membranes (Amersham Inc., Piscataway, NJ). Cyclin D1 probe was generated by subcloning the PCR product obtained from RT-PCR as described above into the pCRII-TOPO vector, linearizing the plasmid with BamHI, and generating a riboprobe using the Riboprobe In Vitro Transcription System as described by the manufacturer (Promega, Inc., Madison, WI). Probe was labeled with 5'-[{alpha}-32P]CTP to a specific activity of 2.0 x 108 cpm/µg. Labeled probe was incubated with membrane at 65°C for 18 h in hybridization buffer (0.1% SDS, 50% formamide, 5x SSC, 50 mM NaPO4 (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt’s solution, and 50 µg/ml salmon sperm DNA) followed by two 5-min washes (1x SSC-0.1% SDS) at 65°C. Membranes were exposed to film for autoradiography.

Data Analysis.
Quantification of bands from kinase assays, Western blots, IPs, RT-PCR, and Northern blots was performed by digital image analysis using a Hewlett-Packard ScanJet 6100C scanner and Gel-Pro Analyzer software (Media Cybernetics, Silver Spring, MD).


   Acknowledgments
 
We thank Joan Boylan for her invaluable advice and assistance in the performance of these studies. We greatly appreciate the work of Theresa Bienieki, who performed the hepatocyte isolations. We also thank the staff in the Central Research Laboratories at Rhode Island Hospital for their assistance in the performance of the flow cytometry studies.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants HD24455 and HD11343 and by the Rhode Island Hospital Department of Pediatrics Research Endowment Fund. Back

2 To whom requests for reprints should be addressed, at Department of Pediatrics, Rhode Island Hospital, 593 Eddy Street, Rhode Island Hospital, Providence, RI 02903. Phone: (401) 444-5504; Fax: (401) 444-2534; E-mail: Philip_Gruppuso{at}brown.edu Back

3 The abbreviations used are: MAP, mitogen-activated protein (kinase); CDK, cyclin-dependent kinase; CKI, CDK inhibitor; IP, immunoprecipitation; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; RT-PCR, reverse-transcription PCR; GST, glutathione S-transferase. Back

4 P. A. Gruppuso, unpublished observations. Back

5 M. M. Awad, H. Enslen, J. M. Boylan, R. J. Davis, and P. A. Gruppuso. Growth regulation via p38 mitogen-activated protein kinase, submitted for publication. Back

Received for publication 1/ 4/00. Revision received 3/17/00. Accepted for publication 4/17/00.


   References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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