C/EBPalpha regulates generation of C/EBPbeta isoforms through activation of specific proteolytic cleavage

Abstract

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP.

FIG. 1

In vitro translation of C/EBPβ mRNA in the presence of C/EBPα wild-type liver extracts leads to an increase in LIP production. (A) Mutation of the first, second, or third ATG in the C/EBPβ 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/EBPβ in the presence of cytoplasmic extracts from C/EBPα wild-type (+/+) or null (−/−) mice. Translation of the wild-type C/EBPβ 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/EBPβ (C-19).

FIG. 2

Nuclear extracts from newborn wild-type livers contain a C/EBPα-dependent specific proteolytic activity that produces C-LIP. (A) In vitro-translated FL is cleaved by nuclear extracts (NE) from livers of C/EBPα wild-type mice (+/+) but not by those from C/EBPα 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/EBPβ cleavage over time in the presence of no extract (−), nuclear extracts from C/EBPα wild-type mice (+/+), or nuclear extracts from C/EBPα null mice (−/−). (C) Densitometric analysis of C/EBPβ cleavage over time (shown as the LIP/LAP ratio). The graph is representative of three independent experiments.

FIG. 3

Proteolytic cleavage of C/EBPβ is specific to the newborn liver. (A) C/EBPβ 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/EBPβ in the tissues shown above. The graph is representative of three or more experiments done with tissues from different animals. NE, nuclear extract.

FIG. 4

Cleavage of C/EBPβ occurs in the liver before birth. (A) Densitometric analysis showing that cleavage is activated in the livers of C/EBPα 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/EBPα null (−/−) livers. The graph represents an average of three independent experiments. NE, nuclear extract. (B) Representative example of a C/EBPβ 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/EBPα null extracts.

FIG. 5

C/EBPα induces specific proteolytic cleavage of C/EBPβ 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/EBPα or with 10 mM glucose (G) as a control. The left and right columns represent two experiments showing that C/EBPα induces specific cleavage of C/EBPβ to generate C-LIP. (First [top] panel) Western analysis for C/EBPβ. 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 β-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/EBPα. (B) Densitometric analysis of three independent experiments shows a two- to fivefold induction of C/EBPβ cleavage with the overexpression of C/EBPα (IPTG-treated cells). Error bars indicate standard deviations.

FIG. 6

Truncated C/EBPβ isoforms generated by cleavage bind to the C/EBP consensus site and form heterodimers with full-length C/EBPβ 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/EBPβ (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/EBPβ (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.

FIG. 7

The protease responsible for the cleavage of C/EBPβ is sensitive to chymostatin. (A) C/EBPβ cleavage assay in the presence of C/EBPα 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/EBPβ cleavage in the absence and presence of the protease inhibitor chymostatin (C). The graph is representative of five independent experiments. +/+, C/EBPα wild-type extracts; −/−, C/EBPα null extracts.

FIG. 8

A calpain protease can cleave C/EBPβ to generate C-LIP. C/EBPβ 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.

FIG. 9

Induction of LIP during liver regeneration occurs by a translational mechanism. (A) In vitro translation of C/EBPβ 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/EBPβ translated from the wild-type C/EBPβ construct (FL), and the right two lanes contain C/EBPβ 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/EBPβ 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/EBPβ 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.