INTRODUCTION

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The CCAAT/enhancer binding proteins (C/EBPs) are transcription factors belonging to the bZIP family of proteins, which are characterized by the presence of a basic region of amino acids involved in DNA binding followed by a leucine zipper motif involved in homo- and heterodimerization with other family members (40). Several C/EBP family members have been described: C/EBPs , , , , , and  (9, 17, 26, 30, 43). C/EBP is highly expressed in liver, adipose tissue, and in certain cells of the lungs and mammary glands (2, 28, 29, 33). It has been shown to be required for hepatic glycogen synthesis and gluconeogenesis; C/EBP^/ mice die shortly after birth due to severe hypoglycemia (41). C/EBP has been shown to play a key role in adipocyte differentiation and lipid accumulation in vitro (5, 45) and in vivo (7, 41) as well as in cellular growth arrest (37, 39). C/EBP is more widely expressed than C/EBP (9). C/EBP^/ mice display several immunity defects (32, 35), and C/EBP has been implicated in the differentiation of myeloid and lymphoid cell lines (32) as well as in adipocyte differentiation (5, 36, 45). C/EBP^/ females are sterile due to a defect in folliculogenesis (34) and exhibit abnormal morphology in the mammary glands (29, 33).

It has been reported that although both C/EBP and C/EBP lack introns, they each can produce several isoforms by the process of alternative translation (1, 10, 19, 25). This process has been suggested to occur through leaky ribosome scanning leading to initiation at downstream AUG codons and yielding several N-terminally truncated products. The production of N-terminally truncated C/EBP isoforms is highly conserved throughout vertebrate evolution (4, 21). The evolutionary similarity suggests that the smaller isoforms have physiological significance, and this suggestion is supported by observations of different functions of the full-length and truncated isoforms of both C/EBP (19, 25) and C/EBP (10, 45). For C/EBP, four putative isoforms have been described: full-length C/EBP (38-kDa FL), 35-kDa LAP (liver-enriched transcriptional activating protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform (1). Due to the N-terminal truncation of the isoforms, both LIP and the 14-kDa isoform lack most of the transactivation domain, and LIP in particular has been proposed to act as a dominant negative inhibitor of C/EBP-mediated transcription (10). There are several lines of evidence that LIP can inhibit the transcriptional activities of LAP and other C/EBPs. Ectopic expression of LAP in 3T3-L1 cells stimulates adipocyte differentiation; however, overexpression of LIP in these cells inhibits preadipocyte conversion (45). This effect most likely occurs through heterodimerization of LIP with the other isoforms of C/EBP or with C/EBP, exerting a dominant negative effect on transcription. It has also been shown that LIP can attenuate the transcriptional activity of LAP at substoichiometric amounts (10). However, a recent report showed that overexpression of LIP in COS-1 cells leads to increased expression of a chloramphenicol acetyltransferase reporter construct containing the C/EBP binding site of the ₁-acid glycoprotein promoter (13). Whether this mechanism involves direct transactivation of this promoter by LIP or an effect on a negative regulator of this promoter has not been determined. Although the targets of C/EBP LAP and LIP isoforms in vivo are not yet fully known, the fact that these isoforms have different functions suggests that the LIP/LAP ratio is important in C/EBP-mediated gene expression.

There are several situations in which the LIP/LAP ratio has been reported to change in the liver. The alteration of this ratio under certain conditions implies that it is regulated and indicates that the LIP/LAP ratio is functionally important for these processes. Examples include the acute-phase response to inflammation (1), liver development (11), and liver regeneration (38). In all of these cases, LIP levels increase transiently, resulting in an increase in the LIP/LAP ratio. In addition, we recently found that newborn C/EBP^/ animals have a greatly altered LIP/LAP ratio in the liver (3). Wild-type newborn livers express high levels of the low-molecular-weight LIP isoform of C/EBP and low levels of the high-molecular-weight LAP isoform. On the contrary, newborn livers from C/EBP^/ animals express a reduced amount of LIP and a high level of LAP. Given these observations, we decided to investigate the mechanism(s) by which C/EBP may regulate C/EBP isoform generation in the liver. In this report, we present evidence for two mechanisms of regulation of the C/EBP LIP/LAP ratio in the liver: a mechanism that is dependent on alternative translation as well as a novel mechanism that is dependent on C/EBP and involves proteolytic cleavage of C/EBP.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Animals and tissue collection. C/EBP^/ (null) mice and wild-type littermates were sacrificed immediately after birth or at the appropriate gestational age. Liver, lung, and brown adipose tissues from the newborn animals were harvested and immediately frozen in liquid nitrogen. Tissues were stored at 80°C until proteins were isolated for analysis.

Isolation of cytoplasmic and nuclear proteins. Nuclei were isolated as previously described (37, 39). In brief, the tissue was homogenized in buffer A (25 mM Tris-HCl [pH 7.5], 50 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 5 mM dithiothreitol [DTT]) containing 100 µM leupeptin, 2 µg of aprotinin per ml, and 1 mM phenylmethylsulfonyl fluoride (PMSF). In some experiments, 50 µM chymostatin was also included in buffer A. (Chymostatin was not included in nuclear extract isolations to be used as a source for the proteolytic cleavage of C/EBP, as it is an inhibitor of this process.) The nuclei were pelleted by centrifugation at 7,000 × g for 10 min and washed once with buffer A. The cytoplasmic extract (supernatant) was stored frozen at 80°C. Nuclear proteins were then isolated by one of two procedures. For isolation of total nuclear protein without extraction, the pelleted nuclei were directly resuspended in an equal volume of sodium dodecyl sulfate (SDS) loading buffer and heated to 95°C for 30 min. For preparation of a nuclear extract by our standard method of high-salt extraction (39), the pelleted nuclei were resuspended in buffer B (25 mM Tris-HCl [pH 7.5], 0.42 M NaCl, 1.5 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, 25% sucrose) containing 100 µM leupeptin, 2 µg of aprotinin per ml, and 1 mM PMSF. This mixture was incubated for 15 min on ice and then centrifuged at 7,000 × g for 10 min. The nuclear extract (supernatant) was stored at 80°C.

Western blot analysis. Western analysis was carried out as previously described (39). Protein (50 to 100 µg) was loaded onto a 12 to 15% polyacrylamide gel containing 0.1% SDS. The proteins were electroblotted onto a nitrocellulose membrane (0.22-µm-pore size; Bio-Rad) after electrophoresis. The blots were blocked with a solution of TBST (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween 20) containing 2% bovine serum albumin and 10% nonfat dry milk for 1 h at room temperature. The blots were then washed several times with TBST plus 1% nonfat dry milk. Conditions for incubation with primary and secondary antibodies were optimized for each antibody. Primary antibodies used for Western analysis included serum specific for C/EBP (polyclonal serum C-19; Santa Cruz Biotechnology), serum specific for the FLAG epitope (polyclonal serum D-8; Santa Cruz), and serum specific for human C/EBP (polyclonal serum B9 [39]). To examine the relative amounts of proteins loaded in the lanes, blots were reprobed with antibodies to -actin (monoclonal serum AC-74; Sigma) after being stripped in a solution containing 100 mM Tris-HCl (pH 7.5), 2% SDS, and 14 mM -mercaptoethanol. Secondary antibodies used were either goat anti-rabbit-horseradish peroxidase (HRP) or goat anti-mouse-HRP antibodies (both from Santa Cruz). Proteins were detected with an ECL kit from Amersham, and Western blots were quantitated with a Molecular Dynamics personal densitometer and Molecular Dynamics ImageQuant software.

In vitro transcription-translation. In vitro transcription-translation of C/EBP and p21 was carried out in the presence of ³⁵S-methionine by use of a TNT reticulocyte lysate kit from Promega. When needed, the translation system was altered by the addition of 0.2 to 1.0 µg of cytoplasmic or nuclear extracts from livers of C/EBP wild-type or null mice or, alternatively, cytoplasmic extracts from regenerating mouse or rat liver. The following constructs were used in this system: pSCT-LAP, pSCT-LAP1, pSCT-LAP2, and pSCT-LAP3 (generous gifts from U. Schibler and P. Descombes) and pBS-p21(sdi1) (39). For more sensitive detection of radiolabeled products, immunoprecipitation of C/EBP was carried out with antibodies specific for the C terminus of C/EBP to detect all isoforms (C-19). Alternatively, antibodies specific for the N terminus of C/EBP were used for detection of the FL isoform only (a gift from S.-C. Lee). For immunoprecipitation of in vitro-translated p21 protein, polyclonal antiserum H-164 (Santa Cruz) was used. Immunoprecipitation reactions were carried out by incubating diluted in vitro-translated proteins (10 mM Tris-HCl [pH 7.5], 125 mM NaCl) with specific antibodies and protein A-agarose overnight at 4°C. Following immunoprecipitation and several washes with buffer, proteins were run on SDS-polyacrylamide gels, transferred to membranes, and visualized by autoradiography.

In vitro cleavage assay. For in vitro cleavage reactions, immunoprecipitated in vitro-translated full-length C/EBP or p21 protein was incubated with 0.2 to 1.0 µg of nuclear extract from C/EBP wild-type or null livers for 30 min at 37°C, unless otherwise indicated. Other cleavage reactions were carried out with the same amount of nuclear extract from developing embryonic liver, newborn lung, newborn brown adipose tissue, or quiescent adult mouse or rat liver tissue. Alternatively, immunoprecipitated C/EBP was incubated with 0.001 to 0.70 U of m-calpain protease (Sigma). Certain cleavage reactions were carried out in the presence of protease inhibitors at the following concentrations: 100 µM leupeptin, 2 µg of aprotinin per ml, 1 mM PMSF, 50 µM chymostatin, 1 µM pepstatin A, 10 mM EDTA, 10 mM EGTA, 10 µM E-64, 10 µg of cathepsin inhibitor I per ml, 3.8 mg of N-acetyl-Leu-Leu-norleucinal (calpain inhibitor I) per ml, 20 mM lactacystin, 300 µM MG132, or 300 µM MG115. Following the incubation, the reaction was stopped by the addition of SDS loading buffer, and the samples were heated to 95°C for 5 min. Proteins were then run on SDS-12 to 15% polyacrylamide gels, transferred to membranes, visualized by autoradiography, and quantitated by densitometry as described above.

Cell culture and transfection. HT-1 cells, which conditionally express C/EBP under the control of the lac repressor system, were previously described (39). The B4 and B5 cell lines are mouse embryonic fibroblasts derived from C/EBP heterozygous or null mice, respectively, by previously described methods (14). Transient transfection was carried out at 30 to 50% confluency with the Fu gene transfection system (Boehringer Mannheim Biochemicals). Cells were transfected with either the wild-type C/EBP construct (FL; described above) or a LIP mutant construct (MT20-FLAG; a generous gift from John Papaconstantinou). For studies involving the effect of C/EBP on the cleavage of C/EBP, HT-1 cells were treated with 10 mM isopropyl--D-thiogalactopyranoside (IPTG) to induce C/EBP at the time of transfection. Control cells were treated with 10 mM glucose. At 24 h after transfection, cells were collected by scraping into phosphate-buffered saline, followed by centrifugation at 400 × g for 5 min. When whole cells were collected, the cell pellet was directly resuspended in an equal volume of SDS loading buffer and heated to 95°C for 30 min. Total nuclear protein was isolated by direct lysis of nuclei in SDS loading buffer as described above.

Protein elution and band-shift assay. To analyze the binding activities of truncated C/EBP isoforms, HT-1 cells were transfected with the C/EBP LIP mutant construct, and C/EBP expression was induced by IPTG (39). Because maximal levels of C/EBP expression in HT-1 cells are observed 8 to 12 h after IPTG stimulation, proteins were isolated 24 h after transfection and IPTG addition. Fractionation of the proteins was carried out as previously described (24). Briefly, 500 to 1,000 µg of protein was separated on an SDS-12% polyacrylamide gel and transferred to a nitrocellulose filter. The membrane was cut into fractions according to molecular weight, and proteins were eluted in 200 to 300 µl of elution buffer (25 mM Tris-HCl [pH 7.5], 50 mM KCl, 1% Triton X-100, 2 mM MgCl, 1 mM DTT). Each elution fraction (5 µl) was used for gel-shift analysis with a bZIP probe containing the C/EBP consensus binding site (44). The conditions for the gel-shift assay were described in our previous publications (39, 44). For the supershift assay, antibodies to C/EBP (C-19) or to the FLAG epitope (D-8) were added to the binding reaction mixture before the probe addition.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Analysis of C/EBP isoform production in a cell-free system. In order to carefully analyze the mechanism(s) of generation of C/EBP isoforms, as well as the putative role of C/EBP in the regulation of this process, we used an in vitro coupled transcription-translation system in reticulocyte lysate. In vitro translation of a wild-type C/EBP construct (10) yielded three products: FL, LAP, and LIP (Fig. 1A, first lane). As expected, LAP and LIP were N-terminally truncated products, since they were immunoprecipitated with antibodies specific to the C/EBP C terminus (see Materials and Methods). The molecular weight of each C/EBP isoform produced in the reticulocyte lysate was consistent with that predicted from the amino acid sequence and was also consistent with the C/EBP of isoforms detected in the liver (3). When the C/EBP construct was not included in the reticulocyte lysate system, no C/EBP-immunoreactive products were produced, ruling out the possibility that endogenous C/EBP mRNAs are responsible for protein production in this system (data not shown). In vitro translation of various C/EBP mutant constructs (10) revealed that mutation of ATG 1, ATG 2, and ATG 3 led to the disappearance of FL, LAP, and LIP, respectively (Fig. 1A). It is apparent that mutation of ATG 2 led to the production of a band with a mobility slightly higher than that of LAP; however, the origin of this band is unknown and has not been investigated in this report. The disappearance of LIP with mutation of ATG 3 indicated that LIP is produced in the reticulocyte lysate system by AUG-dependent translation and not by a mechanism such as proteolysis of C/EBP.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   In vitro translation of C/EBP mRNA in the presence of C/EBP wild-type liver extracts leads to an increase in LIP production. (A) Mutation of the first, second, or third ATG in the C/EBP construct leads to the disappearance of FL, LAP, and LIP, respectively. The positions of the isoforms are indicated at the right. (B) In vitro translation of C/EBP in the presence of cytoplasmic extracts from C/EBP wild-type (+/+) or null (/) mice. Translation of the wild-type C/EBP construct (FL) is shown in the left two lanes, and translation of the ATG 3 (LIP mutant) construct is shown in the right two lanes. The regions of the expected isoforms are indicated at the right, and the doublet for LIP is labeled at the left as C-LIP and LIP (see the text). For these experiments, in vitro-translated products were immunoprecipitated with antibodies specific for the C terminus of C/EBP (C-19).

Addition of extracts from C/EBP wild-type livers, but not from C/EBP null livers, increases LIP production in a cell-free system. To investigate the mechanism(s) of C/EBP isoform generation in the liver, we examined whether nuclear or cytoplasmic proteins present in the newborn liver could influence C/EBP isoform generation. Since very little LIP is observed in nuclei from livers of newborn C/EBP^/ mice compared with their wild-type littermates (3), we investigated whether a factor present in the wild-type newborn liver cytoplasm could increase the generation of LIP. In vitro translation of C/EBP was carried out in the presence of cytoplasmic extracts from C/EBP null or wild-type livers. C/EBP translation in the presence of wild-type cytoplasmic extracts led to higher levels of LIP than were produced in the presence of null cytoplasmic extracts (Fig. 1B, first and second lanes). Subsequently, we determined that the LIP-generating activity was even more pronounced with nuclear extracts. Due to the increased activity in nuclear extracts relative to cytoplasmic extracts, further experiments were carried out with nuclear extracts.

/

FL and LAP can be proteolytically cleaved to generate C-LIP. To determine if the C/EBP-dependent increase in LIP production was due to proteolytic cleavage of C/EBP, we first carried out in vitro translation of C/EBP and then immunoprecipitated the product with antibodies specific for the C/EBP N terminus (18). FL and a small amount of LAP were the only isoforms detectable by this method (Fig. 2A, lane 1, and data not shown). When the immunoprecipitated products were incubated with liver extracts from wild-type newborn mice, proteolytic cleavage of the high-molecular-weight isoforms occurred, yielding a LIP-like molecule as a major cleavage product (C-LIP) as well as two additional cleavage products, C-32 and C-13 (Fig. 2A). Both FL and LAP could be substrates for the specific proteolytic cleavage. When compared directly, the mobility of the LIP isoform produced by translation in the in vitro system was very similar to that of C-LIP generated by proteolysis (Fig. 1B). Because the mobilities of LIP and C-LIP were also very similar when detected with antibodies specific for the C terminus of C/EBP (see below), it is likely that the site of cleavage was close to the methionine residue which would mark the N terminus of LIP. The pattern of cleavage products generated from FL immunoprecipitated following translation was identical to the pattern of isoforms detected with the addition of liver extracts directly to the in vitro translation system (compare Fig. 1B and 2A) and also matched the pattern of isoforms detected in vivo (3). Invariably, proteolytic activity was absent in C/EBP null liver extracts (more than five animals were tested).

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Nuclear extracts from newborn wild-type livers contain a C/EBP-dependent specific proteolytic activity that produces C-LIP. (A) In vitro-translated FL is cleaved by nuclear extracts (NE) from livers of C/EBP wild-type mice (+/+) but not by those from C/EBP null mice (/). Three major cleavage products are observed: C-32, C-LIP, and C-13 (shown at the left). On the contrary, the stability of in vitro-translated p21 protein is not affected by incubation with either wild-type extracts or null extracts. (B) Representative experiment showing an analysis of the relative amount of C/EBP cleavage over time in the presence of no extract (), nuclear extracts from C/EBP wild-type mice (+/+), or nuclear extracts from C/EBP null mice (/). (C) Densitometric analysis of C/EBP cleavage over time (shown as the LIP/LAP ratio). The graph is representative of three independent experiments.

The proteolytic pathway of LIP generation is specific for prenatal and newborn livers. C/EBP is a tissue-specific transcription factor, high levels of which are detected in liver, lung, and adipose tissues (2). We tested nuclear extracts from these tissues for the ability of C/EBP to regulate the proteolytic cleavage of C/EBP (Figure 3). Newborn mouse liver was the only tissue tested in which significant proteolytic cleavage of in vitro-translated C/EBP was detected. Even though C/EBP is expressed at high levels in quiescent mouse and rat liver tissues and in differentiated adipose tissue (2, 38), the factor which specifically cleaves C/EBP is not activated in these tissues. This result is supported by the fact that LIP/LAP ratios are not altered in lung and brown adipose tissues in C/EBP^/ mice (3). These observations suggested that C/EBP expression is necessary but not sufficient for the regulation of LIP production by specific cleavage and that temporal control during liver development is likely to play a role in the regulation of this activity.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Proteolytic cleavage of C/EBP is specific to the newborn liver. (A) C/EBP cleavage assay with no nuclear extract and nuclear extracts from newborn (newb.) liver, adult liver, newborn lung, and newborn brown adipose tissue (BAT). All of these tissues were from wild-type mice. (B) Densitometric analysis of the relative amount of cleavage of C/EBP in the tissues shown above. The graph is representative of three or more experiments done with tissues from different animals. NE, nuclear extract.

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Cleavage of C/EBP occurs in the liver before birth. (A) Densitometric analysis showing that cleavage is activated in the livers of C/EBP wild-type (+/+) mice at the following times during development: 16 days of gestation (16 d), 18 days of gestation (18 d), and newborn animals (newb). No cleavage was detected in C/EBP null (/) livers. The graph represents an average of three independent experiments. NE, nuclear extract. (B) Representative example of a C/EBP cleavage assay with tissues from wild-type mice at the same times during development. The positions of LAP, C-LIP, and C-13 are shown. N, newborn animals; KO, C/EBP null extracts.

C/EBP induces proteolytic cleavage of C/EBP to generate C-LIP in cultured cells. To further investigate the mechanisms of C/EBP isoform generation, as well as the role of C/EBP in this process, we used a cell line in which both LAP and LIP are expressed and in which C/EBP expression can be controlled. The HT-1 cell line is a stable clone (derived from human fibrosarcoma line HT1080) which conditionally expresses C/EBP under the control of the lac repressor system. Cells treated with IPTG are induced to express C/EBP at high levels, whereas glucose-treated control cells express little or no C/EBP (39). We transfected HT-1 cells with wild-type or mutant C/EBP constructs and added glucose or IPTG to the media at the time of transfection. To distinguish the mechanisms of cleavage and translation, we used the MT20-FLAG construct bearing an ATGTTG mutation at the LIP translation initiation site as well as a C-terminal FLAG epitope (13). Western blot analysis with antibodies specific for the C terminus of C/EBP showed that an immunoreactive band (C-LIP) generated by the mutant construct was present in nuclei of transfected cells (Fig. 5A, MT20). This band migrated with a mobility slightly lower than that of LIP produced from other constructs, partially due to the C-terminal FLAG epitope. Endogenous C/EBP did not contribute to the C/EBP isoforms observed here, as it was below the level of detection in these experiments (Fig. 5A, no DNA). Reprobing the same filter with antibodies to the FLAG epitope confirmed that this band was an N-terminally truncated protein specific for the MT20 C/EBP construct. Since this construct had no ATG for LIP production by translation, our data verified that C-LIP can be generated in cultured cells by an ATG-independent mechanism. This observation was in agreement with our finding that C/EBP can be proteolytically cleaved in vitro to generate C-LIP. All of these experiments were carried out by direct lysis of whole nuclei (isolated with chymostatin; see below) in SDS loading buffer, indicating that proteolysis of C/EBP occurs within the cells and not during the preparation of nuclear extracts. Additionally, cleavage of C/EBP to generate C-LIP was also observed in nuclear extracts, whole nuclei, and whole-cell preparations of mouse embryonic fibroblast cell lines derived from C/EBP heterozygous or null animals (B6 and B7 lines, respectively; data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 5.   C/EBP induces specific proteolytic cleavage of C/EBP in cell cultures. (A) Western analysis of HT-1 whole nuclei. Cells were transfected with the wild-type (LAP FL) or the LIP mutant (MT20) construct. At the same time, cells were treated with 10 mM IPTG (I) to induce the expression of C/EBP or with 10 mM glucose (G) as a control. The left and right columns represent two experiments showing that C/EBP induces specific cleavage of C/EBP to generate C-LIP. (First [top] panel) Western analysis for C/EBP. The positions of LIP (arrow) and C-LIP (arrowhead) are shown in the center. C-LIP is slightly shifted, partially because of the presence of a FLAG epitope on the C terminus of the MT-20 construct. (Second panel) The same filter was reprobed with antibodies to FLAG. Again, the position of C-LIP is shown with an arrowhead. (Third panel) The same filter was reprobed again with antibodies to -actin as a control for protein loading. (Fourth [bottom] panel) The same samples were loaded onto a second gel, and the filter was probed with antibodies specific for C/EBP. (B) Densitometric analysis of three independent experiments shows a two- to fivefold induction of C/EBP cleavage with the overexpression of C/EBP (IPTG-treated cells). Error bars indicate standard deviations.

C-LIP molecules generated by proteolytic cleavage bind to DNA and are able to form heterodimers with C/EBP family members. C-LIP molecules are C-terminal cleavage products of C/EBP, since they interact with antibodies specific for the C terminus of C/EBP. Like LIP, C-LIP presumably contains both the DNA binding domain and the leucine zipper region but lacks the transactivation domain due to N-terminal truncation. To examine whether C-LIP binds to the C/EBP consensus site and whether C-LIP forms heterodimers with FL and LAP, HT-1 cells were transfected with the MT20-FLAG construct. As described above, this construct cannot produce LIP molecules by translation but is able to generate the cleavage product, C-LIP. Whole nuclei were isolated from cells 24 h after transfection, and the proteins were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose filter. The membrane was cut crosswise in fractions on the basis of molecular weight, and proteins were eluted as described previously (24). Separation of the C/EBP proteins was examined by Western blotting with antibodies to C/EBP (C-19). Figure 6A shows that FL and LAP molecules were located in fractions 4 and 5, corresponding to 35 to 46 kDa. The truncated isoform generated by cleavage (C-LIP) was detected in fractions 8 and 9, corresponding to 21 to 24 kDa. These isoforms were designated C-LIP1 and C-LIP2. The presence of two cleavage products was in agreement with the pattern of cleavage activity in nuclear extracts from the developing liver (Fig. 4B). A 13- to 14-kDa band was also observed (see below).

View larger version (45K): [in this window] [in a new window]   FIG. 6.   Truncated C/EBP isoforms generated by cleavage bind to the C/EBP consensus site and form heterodimers with full-length C/EBP isoforms. (A) Western analysis of elution fractions containing proteins having different molecular masses. HT-1 cells were transfected with the MT20-FLAG construct, and nuclear proteins were separated on the basis of molecular mass as described in Materials and Methods. Each fraction (shown on the top) was analyzed by SDS-12% PAGE with antibodies to C/EBP (C-19). (B) Each fraction (5 µl) was analyzed by a gel-shift assay as described in Materials and Methods. (C) Gel-shift analysis of the C-LIP molecule. Antibodies (Ab) to C/EBP (C-19) or to the FLAG epitope (FL.) were added to the binding reaction mixture before the addition of the bZIP probe. Positions of supershifted complexes (S) and LAP-C-LIP1 heterodimers are indicated.

C/EBP-dependent cleavage of C/EBP is a result of activation of a protease that is sensitive to chymostatin. To characterize the proteolytic activity present in wild-type liver extracts, we carried out cleavage assays of C/EBP in the presence of several protease inhibitors. Results from these studies showed that complete inhibition of C/EBP proteolysis was achieved with chymostatin, a serine and cysteine protease inhibitor (Fig. 7). We also observed inhibition of C/EBP cleavage with relatively high concentrations of LLnL, also referred to as calpain inhibitor I (3.8 mg/ml; data not shown). No significant inhibition was observed with the protease inhibitors leupeptin, aprotinin, PMSF, pepstatin A, E-64, cathepsin inhibitor I, MG132, MG115, and lactacystin (Fig. 7 and data not shown). This result indicated that inhibitors of lysosomal proteases and specific inhibitors of the proteasome are not effective in protecting C/EBP from cleavage. The addition of either EDTA or EGTA also did not inhibit the proteolytic activity (data not shown). These results indicated that C/EBP is cleaved by a C/EBP-dependent protease that is sensitive to chymostatin and, to a lesser extent, to LLnL. To further characterize the protease activity, we carried out cleavage assays of C/EBP under several different sets of conditions. Although the protease was active under a range of pH conditions, optimal activity was observed at pH 7.5 to 8.8 (data not shown). At pH 7.5, the cleavage activity was not detectable at 4°C but was substantial from 25 to 55°C. Incubation of the extract at 65°C or higher eliminated all activity (data not shown). Proteolytic activity was also stable over a range of salt conditions, with optimal activity at 250 to 400 mM NaCl (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 7.   The protease responsible for the cleavage of C/EBP is sensitive to chymostatin. (A) C/EBP cleavage assay in the presence of C/EBP wild-type or null nuclear extracts (NE) and protease inhibitors (Inhib). First lane, no extract or inhibitor added. Second lane, wild-type extract only. Third lane, null (KO) extract only. Fourth lane, wild-type extract plus 10 µM E-64. Fifth lane, wild-type extract plus 50 µM chymostatin (C). (B) Densitometric analysis of C/EBP cleavage in the absence and presence of the protease inhibitor chymostatin (C). The graph is representative of five independent experiments. +/+, C/EBP wild-type extracts; /, C/EBP null extracts.

View larger version (55K): [in this window] [in a new window]   FIG. 8.   A calpain protease can cleave C/EBP to generate C-LIP. C/EBP cleavage assay in the presence of increasing amounts of the 80K subunit of m-calpain. First lane, no protease added. Second lane, wild-type liver nuclear extract. Third through ninth lanes, 0.001, 0.003, 0.009, 0.025, 0.08, 0.20, and 0.70 U of m-calpain, respectively. LAP and cleavage products are labeled at the left.

The C/EBP LIP isoform is generated by a translation-dependent mechanism during liver regeneration. In this report, we have shown with several different systems that C/EBP is involved in the activation of a pathway of cleavage, possibly through the activation of a calpain protease, that leads to the conversion of high-molecular-weight isoforms of C/EBP to C-LIP. However, our experiments with cell cultures indicated that a translational mechanism for generating LIP also exists (Fig. 5A) and is, in fact, the major mechanism in these cell lines. LIP generation by translation is also in agreement with other reports (10, 13). Therefore, we examined whether there are other situations in the liver in which the upregulation of LIP occurs by a translation-dependent mechanism. Since we can distinguish the processes of translation and cleavage in vitro by using the LIP mutant construct, we tested another system in which C/EBP isoforms are altered: the process of liver regeneration. C/EBP is required for normal liver regeneration following partial hepatectomy (PH) (12). During the regeneration process, there is a transient increase in LIP production, leading to an increase in the LIP/LAP ratio (38).

View larger version (49K): [in this window] [in a new window]   FIG. 9.   Induction of LIP during liver regeneration occurs by a translational mechanism. (A) In vitro translation of C/EBP in the absence () or presence of cytoplasmic extracts (CE) from regenerating rat livers. Extracts were added from rat livers at 0 or 6 h (HRS) after PH (times indicated above the gel). The left three lanes contain C/EBP translated from the wild-type C/EBP construct (FL), and the right two lanes contain C/EBP translated from the LIP mutant construct (ATG 3). The positions of LAP and LIP are indicated at the right. (B) In vitro translation of C/EBP in the presence of CE from regenerating mouse livers. Extracts were added from mouse livers at 0, 3, or 36 h after PH (times indicated above the gel). CE from two different animals were used in parallel. The left column depicts the use of the wild-type C/EBP construct (FL), and the right column depicts the use of the LIP mutant construct (ATG 3). The positions of LAP and LIP are indicated at the right.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

That several protein isoforms can be generated from a single gene through the process of alternative splicing has been well described (for a recent review, see reference 8). However, some intronless genes produce different protein isoforms from a single mRNA species. In this paper, we provide evidence for two specific mechanisms for the production of the low-molecular-weight C/EBP isoform LIP. The first mechanism is dependent on translation initiation at the third AUG codon in C/EBP mRNA. This mechanism seems to be the major pathway by which LIP is generated in the cultured cell lines that we tested, since mutation of this ATG greatly reduced the amount of LIP produced. Similarly, mutation of this ATG completely inhibited the upregulation of LIP seen with the addition of cytoplasmic extracts from regenerating liver to an in vitro translation system. Our observations with both of these systems verified that LIP can be generated via translation initiation at the third AUG codon of C/EBP. Generation of the LIP isoform by translation is also in agreement with other reports in which C/EBP isoforms were examined with cultured cells (10, 13).

Our data showed that LIP can also be generated by a second mechanism: C/EBP-dependent proteolytic cleavage of FL and LAP. We designated this product C-LIP to distinguish it from the LIP product that is generated by the translation-dependent mechanism. LIP and C-LIP have virtually indistinguishable properties. Like LIP, C-LIP is an N-terminally truncated isoform; they have similar molecular weights. Furthermore, C-LIP can form heterodimers with other C/EBP family members and bind to the C/EBP consensus site. For these reasons, it is likely that the function of C-LIP is equivalent to that of LIP. We also determined that the generation of C-LIP by specific proteolytic cleavage of C/EBP occurs within cells and is very unlikely to result from the isolation procedure, as was recently proposed (21).

We examined the proteolytic pathway of C-LIP generation and found that a key component of this mechanism is a dependence on C/EBP. In several different systems, C/EBP contk;1trolled production of the C-LIP molecule. First, C/EBP^/ mice expressed reduced levels of LIP in the liver compared to their wild-type littermates (3). Second, extracts from C/EBP wild-type livers but not C/EBP null livers could cleave C/EBP to produce C-LIP in vitro. Third, overexpression of C/EBP in cell cultures led to increased cleavage of FL and LAP to produce C-LIP. We also found that the specific cleavage activity occurred in the prenatal liver when C/EBP was expressed during development. This correlation was consistent with the observation that C/EBP regulated this process. Although C/EBP is highly expressed in several tissues, including liver, lung, and adipose tissues (2, 38), the cleavage activity was found to be quite specific for prenatal and newborn livers. This finding suggested that C/EBP is necessary but not sufficient for activation of the cleavage of C/EBP and that regulation of the protease responsible for this cleavage may require other transcription factors that are present in neonatal and newborn livers. The specificity of the cleavage activity for the newborn liver suggested that C/EBP isoforms have important roles during development. It is interesting that no cleavage activity was detectable in quiescent adult liver, despite high levels of C/EBP in this tissue (38). Whether the cleavage of C/EBP leading to an increase in LIP production is involved in the proliferation of hepatocytes in the developing neonatal liver remains to be determined. However, high levels of LIP have been associated with mammary neoplasias and may contribute to cellular proliferation (27). Unlike the mechanism of alternative translation, the cleavage mechanism simultaneously decreases levels of LAP and FL while increasing levels of LIP. It is possible that these activities constitute fine-tuning of C/EBP-mediated transcription in the newborn liver. We also demonstrated that an approximately 13-kDa isoform of C/EBP can be generated by cleavage. This isoform may be similar to the previously reported 14-kDa isoform of C/EBP (1). We showed that this molecule can bind to DNA; however, because of N-terminal truncation, this isoform lacks the transactivation domain and may have a dominant negative function similar to that of LIP.

Identification of in vivo targets of all of these isoforms will aid in understanding the importance of the specific cleavage of C/EBP. The fact that this mechanism is controlled by another C/EBP family member, C/EBP, implies a possible feedback mechanism for balancing transcriptional control in the newborn liver. It is also interesting to note that during liver regeneration following PH, when we observed only translation-dependent upregulation of LIP, C/EBP was reduced to low levels (38). This finding correlated with the lack of C/EBP-dependent cleavage activity at this time.

Although the activation of various transcription factors by posttranslational modification such as phosphorylation or dephosphorylation has been well investigated, little has been described about the regulation of transcription factors by proteolytic processing. One of the most well-characterized pathways in which specific proteolytic processing plays a major role in regulating the activity of a transcription factor is the NF-B pathway. The p50 subunit is generated from a p105 precursor by a ubiquitin-mediated proteolytic processing event in the cytoplasm of most cells (6), and it has been reported that this processing event is cotranslational (20). Additionally, the NF-B complex (p50-p65) is sequestered in the cytoplasm by inhibitory IB proteins, and it is the proteolytic degradation of IB that releases NF-B proteins for subsequent nuclear localization and activation of target genes (22, 23, 31). It has been reported that targeted proteolysis of the nuclear receptor corepressor N-CoR is a cell-specific mechanism for regulating gene transcription (15). Although both of these pathways involve the regulation of transcriptional activators or repressors by pathways of proteasomal degradation, we report in this paper that the specific cleavage of C/EBP is not prevented by inhibitors of the proteasome, such as lactacystin, MG132, and MG115. This result suggested that the C/EBP-dependent cleavage of C/EBP occurs by a mechanism independent of the proteasome. Nonproteosomal cleavage of transcription factors has also been reported to occur. For example, the bHLHzip factor USF, as well as a number of other transcription factors, is specifically cleaved by m-calpain, leaving the DNA binding and dimerization domains intact. The resultant protein can bind to DNA but can no longer activate transcription (42). In this report, we show that the cleavage of C/EBP also yields a product, C-LIP, that can bind to DNA but that lacks a transactivation domain. Taken together, these results suggest that targeted proteolysis of certain transcription factors may be an important mechanism for rapidly regulating the expression of target genes.

Although the enzyme responsible for C/EBP cleavage has not yet been purified, we have determined that C-LIP is generated by a protease that is sensitive to both chymostatin and LLnL. We have also shown that a commercially available preparation of m-calpain can cleave C/EBP to generate C-LIP. These data suggested that the C/EBP-dependent cleavage of C/EBP may be due to the activation of a calpain protease in liver. Whether the proteolytic activity in the newborn liver is identical to that found in the calpain preparation is currently under investigation. The purification and identification of the enzyme from liver will provide a greater understanding of the mechanism of cleavage, as well as the regulation of the protease activity by C/EBP.