Immunological characterization of tristetraprolin as a low abundance, inducible, stable cytosolic protein

Abstract

Tristetraprolin (TTP) is a zinc finger protein that can bind to AU-rich elements within certain mRNAs, resulting in deadenylation and destabilization of those mRNAs. Its physiological targets include the mRNAs encoding the cytokines tumor necrosis factor alpha (TNF) and granulocyte-macrophage colony-stimulating factor. TTP was originally identified on the basis of its massive but transient increase in mRNA levels following mitogen stimulation of fibroblasts. It has been difficult to reconcile this transient mRNA profile with the presumed continuing "need" for TTP protein, for example, to reverse the effects of lipopolysaccharide (LPS)-stimulated TNF secretion. To investigate this and other questions concerning endogenous TTP protein in cells and tissues, we raised a high titer rabbit antiserum against full-length mouse TTP. TTP could be detected on immunoblots of mouse cytosolic tissue extracts; it was most highly expressed in spleen, but its concentration in that tissue was only about 1.5 nm. TTP could be detected readily in splenic macrophages and stromal cells from LPS-injected rats. In both LPS-treated RAW 264.7 macrophages and fetal calf serum-treated mouse embryonic fibroblasts, TTP protein was stable after induction, with minimal degradation occurring for several hours after treatment of the cells with cycloheximide. The biosynthesis of TTP was accompanied by large changes in electrophoretic mobility consistent with progressive phosphorylation. Confocal microscopy revealed that TTP accumulated in a vesicular pattern in the cytosol of the LPS-stimulated RAW 264.7 cells, and was occasionally seen in the cytosol of unstimulated dividing cells. Gel filtration of the endogenous protein suggested that its predominant structure was monomeric. TTP appears to be a low abundance, cytosolic protein in unstimulated cells and tissues, but once induced is relatively stable, in contrast to its very labile mRNA.

Fig. 1

Purification of MBP-mTTP from E. coli and characterization of antibodies. MBP-mTTP was purified from E. coli transformed with plasmid pMBP-mTTP. MBP-mTTP was initially purified from a 10,000 × g supernatant by amylose resin chromatography, followed by Superose 12 size exclusion and Mono Q anion exchange chromatography. A, purification of MBP-mTTP stained with Coomassie Blue: lane 1, protein size standards; lane 2, homogenate (50 μg of protein); lane 3, supernatant (50 μg); lane 4, amylose resin column peak fraction (5 μg); lane 5, Superose 12 column peak fraction (1 μg); lane 6, Mono Q column peak fraction (1 μg); and lane 7, MBP eluted from the amylose resin column (5 μg). The positions of MBP-mTTP and MBP are indicated. B, detection of MBP-mTTP and MBP by anti-MBP serum. The samples were identical to those shown in A except that about 10% of the amount of protein was used in each lane. C, characterization of anti-MBP-mTTP serum by Western blotting. The indicated amounts of MBP-mTTP eluted from the amylose resin column, and HA-mTTP in soluble extracts of transfected 293 cells, were probed with the anti-MBP-mTTP serum (1:10,000) and GAR-HRP (1:10,000) for 30 min each before being incubated in the detection reagent for 5 min and exposed to x-ray film for 30 s. The smaller immunoreactive bands seen both with the E. coli protein and the protein expressed in 293 cells are presumed to be proteolytic fragments.

Fig.2

Identification of endogenous TTP in mouse spleen. A, 2 μg of protein from 293 cell extracts expressing HA-mTTP, and 5 mg of protein from 20,000 × g supernatants of spleen homogenate from WT and TTP KO mice, were separated by SDS-PAGE and transferred onto nitrocellulose membranes for Western blotting. The membrane was incubated in anti-MBP-mTTP serum (1:10,000) overnight and with GAR-HRP (1:10,000) for 1 h and exposed to x-ray film for 1 min. B, the 20,000 × g supernatant from mouse spleen was further centrifuged at 100,000 × g for 1 h to separate microsomal membranes (Membranes) from the cytosol (Soluble). Equal amounts of protein (1.7 mg) from the soluble and membrane fractions were used for Western blotting as above, along with MBP-mTTP (10 ng) from the amylose resin column and HA-mTTP in the transfected 293 cell extracts (1 μg of total soluble protein). In this case, the membrane was exposed to x-ray film for 10 min.

Fig. 3

Expression of TTP in mouse tissues. A supernatant (10,000 × g) was prepared from tissues from WT and TTP KO mice and was used for immunoblotting. Transfected 293 cell extracts expressing HA-mTTP (1 μg of total protein) were used as positive controls. A, in each lane, 1.7 mg of total protein in the supernatant from WT mouse tissue was used, and the blot was exposed to x-ray film for 4 min. The broad band or doublet corresponding to TTP is indicated. B, TNFR1 KO (TTP WT); C, TNFR1/TTP double knockout (TTP KO) tissue extracts. 1 mg of total protein in the tissue supernatants was used, and the blots were exposed to x-ray film for 1 h in the same cassette. The bands seen in B but not in C represent specific TTP immunoreactivity; the bands shown in both blots represent nonspecific protein reactivity.

Fig.4

TTP expression in primary mouse macrophages, RAW 264.7 cells, and MEF. A, primary mouse macrophages from TTP WT and KO mice were treated with LPS (1 μg/ml) for 2 h, and soluble cellular extracts were prepared. Equal amounts of protein from these extracts (200 μg) were used for immunoblotting, using the anti-MBP-mTTP serum (1:10,000). The blot was exposed to x-ray film for 30 s. The position of immunoreactive TTP is shown in the WT but not the KO cells. B, RAW 264.7 cells and MEF. RAW 264.7 cells (RAW) were stimulated with LPS (0.1 μg/ml) for 1.5 and 2 h, and used for the preparation of 10,000 × g supernatants. Similarly, MEF from WT and TTP KO mice were stimulated with 10% FCS for 2 and 3 h. Supernatants from RAW (50 μg) and MEF (500 μg) were used for immunoblotting with the anti-MBP-mTTP serum (1:10,000). The blot was exposed to x-ray film overnight. The position of TTP is indicated; note its absence in the KO MEF.

Fig. 5

Time course of induction and stability of TTP in mouse cells. RAW 264.7 cells and primary mouse macrophages were stimulated with 0.1 μg/ml LPS, and MEF were stimulated with 10% FCS, for various times as indicated. To measure protein stability, the cells were stimulated for 2 h before being treated with CHX (50 μM) for the indicated times. A supernatant (10,000 × g) was prepared from the cells and used for immunoblotting using the anti-MBP-mTTP serum (1:10,000). A, data from RAW 264.7 cells (RAW) (50 μg of protein per lane) were compared with data from primary macrophages (500 μg per lane). The blot was incubated in the primary antiserum for 2 h and the secondary antibody for 1 h, then exposed to x-ray film overnight. B, data from RAW 264.7 cells stimulated by LPS for 2 h (10 μg per lane) were compared with data from MEF (fibroblasts) stimulated with FCS with or without the later addition of CHX (200 μg of protein per lane). The blot was incubated in the primary antiserum overnight and the secondary antibody for 2 h, then exposed to x-ray film for 2 min. C, time course of TTP induction in RAW 264.7 cells (50 μg of protein per lane). The blot was incubated in the primary antiserum for 18 h and the secondary antibody for 1 h. The blot was exposed to x-ray film for 5 s. D, time course of TTP induction in RAW 264.7 cells without CHX (50 μg of protein per lane). The blot was incubated in the primary antiserum for 1.5 h and the secondary antibody for 1 h. The blot was exposed to x-ray film for 30 s. E, TTP stability in RAW 264.7 cells (50 μg of protein per lane). The cells were first stimulated with LPS for 2 h, followed by treatment with CHX for the indicated times. The blot was incubated in the primary antiserum for 1.5 h and the secondary antibody for 1 h, then exposed to x-ray film for 30 s.

Fig. 6

Size exclusion chromatography and dephosphorylation of endogenous TTP from RAW 264.7 cells. A, proteins in the 10,000 × g supernatant fraction from RAW 264.7 cells following LPS induction and ammonium sulfate concentration were separated by size exclusion chromatography with a Superose 6 column. TTP was detected by immunoblotting with the anti-MBP-mTTP antiserum (1:10,000). The position of TTP is indicated. B, the size of TTP was determined with a standard curve generated with protein standards separated on the same column under the identical conditions, in which K_av = (V_e−V_o)/(V_t−V_o), where V_e, V_o, and V_tare the elution volume of the protein determined by the experiment, the void volume determined with blue dextran, and the bed volume of the column provided by the manufacturer, respectively. The peak fraction containing TTP (#31) corresponded to a molecular size of 40 kDa (diamond). C, cytosolic localization of TTP in RAW 264.7 cells. RAW 264.7 cells were fractionated into cytosolic and nuclear fractions in two separate experiments after stimulation with LPS (0.1 μg/ml) for 4 and 6 h. Equal amounts of protein from the cytosolic and nuclear fractions (50 μg) were used for immunoblotting with anti-MBP-mTTP serum (1:10,000). Human TTP (no fusion partner) purified from E. coli (20 ng) was used to demonstrate the electrophoretic mobility shift of TTP in RAW 264.7 cells following LPS induction. This blot was incubated in the primary antiserum for 1.5 h and the secondary antibody for 1 h. The blot was exposed to x-ray film for 10 s. D, dephosphorylation of mTTP in LPS-stimulated RAW 264.7 cell extracts. RAW 264.7 cells were stimulated with LPS (0.1 μg/ml) for 0, 1, 2, 3, 4, and 6 h. The 10,000 × g supernatant (100 μg of total protein) was incubated with or without CIAP (35 units) at 23°C (room temperature) for 1 h. Half of the mixture was used for immunoblotting detection. This blot was incubated in the primary antiserum for 18 h and the secondary antibody for 1 h. The blot was exposed to x-ray film for 10 s. “hTTP” refers to the same protein as in C, lane 5, above.

Fig. 7

Confocal microscopy detection of TTP in RAW 264.7 cells. A, confocal microscopy of RAW 264.7 cells. Cells were treated with either LPS (0.1 μg/ml) or PBS for 3 h, then fixed and stained with either the anti-MBP-mTTP serum (I) or preimmune serum (PI) (1:8,000 dilution). Confocal exposures and image processing were the same for all four panels. B, cytosolic localization of TTP in RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS (0.1 μg/ml) for 2 h and stained with anti-MBP-mTTP serum (1:10,000 dilution) as described in A. Serial optical sections of the same cell were collected at 0.5-μm intervals; the upper left image is approximately in the middle of the cell, whereas the bottom right image is closest to the glass surface. C, time course of TTP induction in RAW 264.7 cells. RAW 264.7 cells were stimulated with LPS (0.1 μg/ml) for 0, 2, 3, and 5 h as indicated and stained with anti-MBP-mTTP serum (1:10,000 dilution) as described in A. D, TTP immunostaining during cell division in RAW 264.7 cells. Unstimulated RAW 264.7 cells were stained with the anti-MBP-mTTP serum (1:10,000 dilution), as described in A. Immunoreactive TTP was visible in the cytoplasm of the dividing cell indicated by the arrowheads in D, panel 1, whereas no cytoplasmic staining was visible in the other cells in the field. A light microscopic image of the same field of cells is also shown (D, panel 2).

Fig.8

TTP immunostaining in rat spleen. Rats were injected intraperitoneally with LPS (5 mg/kg; A and B) or PBS (5 ml/kg, C and D), and spleens were removed and used for immunostaining of TTP with either the anti-MBP-mTTP serum (I) or pre-immune serum (PI) as described under “Materials and Methods.” The reddish brown staining in A represents TTP immunoreactivity, whereas none was seen in the other three panels. The arrows in A point to the white pulp of the spleen, which is unstained by the TTP antibody. See the text for other details.