Induction of extracellular matrix-remodeling genes by the senescence-associated protein APA-1

Abstract

Human fibroblasts undergo cellular senescence after a finite number of divisions, in response to the erosion of telomeres. In addition to being terminally arrested in the cell cycle, senescent fibroblasts express genes that are normally induced upon wounding, including genes that remodel the extracellular matrix. We have identified the novel zinc finger protein APA-1, whose expression increased in senescent human fibroblasts independent of telomere shortening. Extended passage, telomerase-immortalized fibroblasts had increased levels of APA-1 as well as the cyclin-dependent kinase inhibitor p16. In fibroblasts, APA-1 was modified by the ubiquitin-like protein SUMO-1, which increased APA-1 half-life, possibly by blocking ubiquitin-mediated degradation. Overexpression of APA-1 did not cause cell cycle arrest; but, it induced transcription of the extracellular matrix-remodeling genes MMP1 and PAI2, which are associated with fibroblast senescence. MMP1 and PAI2 transcript levels also increased in telomerase-immortalized fibroblasts that had high levels of APA-1, demonstrating that the matrix-remodeling phenotype of senescent fibroblasts was not induced by telomere attrition alone. APA-1 was able to transactivate and bind to the MMP1 promoter, suggesting that APA-1 is a transcription factor that regulates expression of matrix-remodeling genes during fibroblast senescence.

FIG. 1.

Sequence of APA-1. (A) Schematic diagram of APA-1. APA-1 contains 478 amino acids, with five C₂H₂-type zinc fingers between amino acids 219 and 361 and a leucine zipper between amino acids 404 and 426. (B) Alignment of human and mouse APA-1 proteins. Identical residues are shaded grey. The two sequences are 94% identical.

FIG. 2.

APA-1 expression in human fibroblasts. (A) Increased APA-1 protein in senescent HFFs. Western blot of APA-1 protein in HFFs at increasing population doubling levels (PDLs). Cells were senescent at PDL 76. Protein concentration of each lysate was measured, and 40 μg of total protein was analyzed by Western blotting for APA-1. (B) Constant APA-1 mRNA in HFFs throughout their life span. Total RNA (20 μg) from HFFs at the indicated PDLs were analyzed by Northern blot with probes to APA-1 and 36B4 (loading control). (C) APA-1 and p16 increase in hTert-immortalized fibroblasts. Western blots of 40 μg of total protein in control (LXSN) and hTert-transduced HFFs. Cells were transduced mid-life span. PDL represent population doublings after selection. LXSN cells reached senescence at PDL 44, and hTert-immortalized cells continued proliferating past PDL 109. Lysates were examined for expression of APA-1, p16, and actin. (D) Decreased APA-1 expression in SV40-transformed fibroblasts. Lysates were collected from several normal human fibroblast types (HFF, IMR90, HSF43, and HS74) as well as cells containing SV40 (SV/HF-5, Cl39 p12 and p30, and Cl39T). Total protein (40 μg) was analyzed by Western blot for APA-1 and actin. (E) APA-1 expression in various cell types. Western blot of 40 μg of protein lysate from HFF, HFK, U2OS, and HeLa cells as well as in vitro-translated APA-1 (IVT-APA-1) and a negative control (IVT-H₂O, rabbit reticulocyte lysate alone). The two predominant forms of APA-1 protein run at approximately 67 and 49 kDa and are indicated with arrowheads

FIG. 3.

APA-1 is modified by SUMO-1 in vivo. (A) U2OS transfectants express two forms of APA-1. U2OS cells were transfected with HA-tagged APA-1. After selection, the pool of cells was compared to untransfected U2OS cells and HFFs for expression of APA-1. Two forms of APA-1 are overexpressed in the HA-APA-1 cells (arrowheads), one of which runs close to the 67-kDa form seen in fibroblasts. These were observed by Western blot with both anti-APA-1 and anti-HA antibodies. (B) APA-1 in HFFs is modified by SUMO-1. HFFs were lysed by denaturing lysis in SDS, diluted, and immunoprecipitated (IP) with anti-APA-1 antiserum or preimmune serum (p.i.). Immunoprecipitates were eluted in sample buffer, split in half, and loaded onto two gels. These were Western blotted for APA-1 or SUMO-1. The 67-kDa form of APA-1 was immunoprecipitated and was recognized by both antibodies.

FIG. 4.

Sumoylation increases the half-life of APA-1. (A) Cycloheximide time course comparing the stability of unmodified and sumoylated APA-1. HFFs were transduced with either empty vector (LXSN) or APA-1. After selection, cells were plated in 10-cm dishes and treated with 25 μM cycloheximide to block protein synthesis. Lysates were collected at the indicated time points between 0 and 32 h. Protein concentrations were determined, and 40 μg of each lysate was analyzed by Western blotting for APA-1. Arrowheads represent sumoylated (APA-1-S) and unmodified (APA-1) APA-1 proteins. Approximate protein levels were quantitated with a Fluor-S-Multiimager (Bio-Rad), and the half-life of unmodified APA-1 was determined to be approximately 2 h. (B) Proteasome inhibitors increase levels of unmodified APA-1 in HFFs. HFFs were treated with 25 μM each of the proteasome inhibitors MG132 and ALLN, the calpain inhibitor ALLM, or an equivalent volume of dimethyl sulfoxide (DMSO) for 4 h, and lysates were collected. Then 40 μg of total protein was analyzed for levels of APA-1, p53, and actin. (C) APA-1 is polyubiquitinated in vivo. U2OS cells were transfected with APA-1 and HA-tagged ubiquitin (HA-Ub) or an empty vector control. Denaturing lysates were made, diluted, and immunoprecipitated (IP) with antibodies against HA or APA-1, as indicated. HA immunoprecipitates were analyzed by Western blotting (WB) for APA-1, and APA-1 immunoprecipitates were analyzed by Western blotting for HA. The positions of unmodified APA-1 and immunoglobulin G heavy chain (IgG) are indicated by arrowheads.

FIG. 5.

APA-1 overexpression does not induce senescence. (A) Western blots of transduced HFFs. Mid-life HFFs were infected with concentrated retroviruses expressing empty vector (LXSN), p16, APA-1, or p14^ARF. After selection, lysates were collected and protein concentrations were measured. Then 40 μg of total protein was analyzed by Western blot for expression of p16, APA-1, p14^ARF, p53, p21, and actin. Overexpression of p16, p14^ARF, and APA-1 was confirmed in the corresponding infections. (B) After selection, the cells from A were labeled with 10 μM BrdU for 4 h and fixed in 70% ethanol. Nuclei were labeled with anti-BrdU-fluorescein isothiocyanate and propidium iodide and analyzed by flow cytometry. Percent BrdU-positive cells is presented. (C) The cells from A along with early-passage (HFF PDL 35) and senescent (HFF PDL 75) controls were stained for senescence-associated (SA) β-galactosidase. Percent positive cells is presented.

FIG. 6.

APA-1 overexpression induces transcription of extracellular matrix-remodeling genes. (A) Early-passage HFFs were infected with concentrated retrovirus expressing empty vector (LXSN) or APA-1 and selected as before. APA-1 protein was analyzed by Western blot and compared to uninfected early-passage (early) and senescent HFFs. Arrowheads indicate the two forms of APA-1. (B) APA-1 induces transcription of senescence-associated matrix-remodeling genes. Total RNA was harvested from the cells described in A and analyzed by Northern blotting for MMP1, PAI2, MMP12, MMP2, and 36B4 (loading control). Specific bands are represented by arrowheads, and the asterisk (*) represents residual signal on the PAI2 blot from a previous probe. Signals were quantified by phosphorimaging, and relative signal (with respect to the 36B4 loading control) is indicated beneath each blot. (C) hTert-immortalized fibroblasts have increased levels of MMP1 and PAI2 mRNAs, similar to senescent controls. RNA was harvested from the cells described in Fig. 2D as well as Cl39T cells. Then 30 μg of total RNA was analyzed by Northern blotting for MMP1, PAI2, and 36B4. Relative signals as determined by phophorimaging are indicated beneath each blot.

FIG. 7.

APA-1 transactivates and binds to the MMP1 promoter. (A) Early-passage ARF^−/− mouse embryo fibroblasts (MEFs) were transfected with the indicated reporter plasmid (MMP1-624 or MMP1-1606) and empty vector (pCDNA) or APA-1. Luciferase values were normalized to protein concentration and graphed relative to the pCDNA3 control for the MMP1-624 promoter fragment. Shown is an average of three experiments carried out in triplicate. (B) Schematic diagram of the MMP1 promoter. Boxes indicate previously described TATA (grey) and PEA3 (black) elements, as well as the putative APA-1 binding site (hatched). Overlapping probes (A through E) used for gel shift analysis are indicated. (C) APA-1 binds to the MMP1 promoter. MMP1 gel shift probes B to D were used in binding reactions with control extracts (pCDNA) or extracts expressing HA-APA-1, as indicated. APA-1 antibody (A) or anti-HA antibody (H) only competed away binding of the APA-1-specific shift of probe C. (D) Four-base-pair substitutions disrupt APA-1 binding. Gel shift analysis of probe C with the wild-type sequence (ATATTGGAG) or with mutations in the putative APA-1 binding site (AGATGAGCG, with substitutions shown in outline letters) and control (C) or HA-APA-1 (A)-expressing extracts, as indicated. The APA-1-specific shift is not competed by addition of preimmune antibody (P) but is competed by addition of APA-1 (A) or anti-HA (H) antibodies. (E) APA-1 binding is necessary for transactivation of the MMP1 promoter. ARF^−/− MEFs were transfected with MMP1-624 reporter plasmid or MMP1-624m reporter plasmid containing the same changes as in D and the vector control (pCDNA) or APA-1 expression plasmid. Luciferase values were normalized to protein concentration and graphed relative to the pCDNA3 control for the MMP1-624 reporter. Shown is an average of three experiments carried out in triplicate.

FIG. 8.

Model for induction of matrix-remodeling genes during fibroblast senescence. Although telomere shortening induces the senescent cell cycle arrest in fibroblasts, an additional signal, possibly resulting from an accumulation of stress, leads to upregulation of the p16 and APA-1 proteins. If APA-1 is necessary for induction of matrix-remodeling genes in late-passage cells, this suggests that the matrix-remodeling phenotype is separable from telomere-induced cell cycle arrest during senescence.