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Down-regulation of amphiphysin-1 is responsible for reduced receptor-mediated endocytosis in the senescent cells¹

JEONG-SOO PARK^*,2, WOONG-YANG PARK^*,2, KYOUNG-A CHO^*, DEOK-IN KIM^*, BYUNG-HAK JHUN, SEUNG-RYUL KIM and SANG CHUL PARK^*3

^* Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul;
Department of Pharmacology, Pusan National University College of Pharmacy, Pusan; and
Department of Biochemistry, Chungbuk National University College of Medicine, Cheongju, South Korea

³Correspondence: Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, 28 Yongondong, Chongnogu, Seoul, 110–799, South Korea. E-mail: scpark{at}snu.ac.kr

SPECIFIC AIMS

Human diploid fibroblasts undergo multiple functional alterations during their senescence process. Most of all, senescent cells do not respond properly to external stimuli and do not uptake materials as efficiently as presenescent cells. To elucidate the reason for such attenuated response of senescent cells, we attempted to monitor the functional capacity and molecular mechanism for the clathrin-dependent, receptor-mediated endocytosis in the senescent cells, and tried to find principal molecules responsible for the decreased endocytotic activity in senescent cells that might be a target of intervention for the age-related dysfunctions.

PRINCIPAL FINDINGS

1. Clathrin-dependent receptor-mediated endocytosis is blocked in senescent human diploid fibroblasts
To investigate the functional changes in the receptor-mediated endocytosis during cellular senescence, we treated the early and late-passaged fibroblasts with tetramethylrhodamine-conjugated transferrin. Presenescent fibroblasts took up transferrin in a 5 min pulse treatment and internalized transferrin rapidly disappeared after an additional 5 min incubation of chasing. However, in senescent cells, we found no image of internalized transferrin even after 10 min of chasing. Even after 60 min of continuous incubation, senescent cells showed no evidence of internalization of the fluorescent transferrin. This alteration of receptor-mediated endocytosis was also evident in hydrogen peroxide-induced premature senescent fibroblasts. These data indicate that receptor-mediated endocytosis via clathrin-coated vesicles is significantly deteriorated in the senescent fibroblasts.

2. Amphiphysin-1 protein was down-regulated in the senescent human diploid fibroblasts
To identify the molecular mechanism for such an alteration in the receptor-mediated endocytotic function of senescent cells, the expression level of several proteins associated with receptor-mediated endocytosis in early-, middle-, and late-passaged fibroblasts, such as transferrin receptor, clathrin heavy chain, the and ß subunits of the AP-2 clathrin adapter complex (-adaptin and ß-adaptin, respectively), amphiphysin-1, and dynamin, was checked by Western blot analysis. It was shown that only amphiphysin-1, but none of the other endocytotic proteins tested, was significantly reduced during cellular senescence. Amphiphysin-1 protein was also down-regulated in premature senescent cells induced by hydrogen peroxide treatment. These results suggest that amphiphysin-1 may play a critical role in the alteration of receptor-mediated endocytosis of the senescent fibroblasts.

3. Dominant negative mutant of amphiphysin-1 inhibited receptor-mediated endocytosis in presenescent fibroblasts
To clarify the functional significance of the amphiphysin in receptor-mediated endocytosis, we checked the effect of the functional inhibition of amphiphysin-1 protein in presenescent cells. To inhibit the function of amphiphysin-1 protein, a dominant negative mutant of amphiphysin-1 that encoded the middle portion (amino acids 250–588) containing the AP-2 and clathrin binding sites was transfected into presenescent fibroblasts. After transfection, cellular capacity to internalize the transferrin was monitored with rhodamine-conjugated transferrin. As shown in Fig. 1A , GFP-positive cells that overexpressed the dominant negative mutant of amphiphysin-1 protein could not uptake transferrin as well as neighboring nontransfected cells. However, mock-transfected cells revealed no functional alteration in uptake of transferrin (Fig. 1B ). In this experiment, we clearly demonstrated that the functional incompetence of amphiphysin-1 could inhibit receptor-mediated endocytosis in human diploid fibroblasts.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of dominant negative mutant amphiphysin-1 protein in presenescent HDFs. A) Presenescent fibroblasts were transfected with pEGFP-N1(mock) or pDN-amph-1-EGFP (DN-amph-1-GFP). At 36 h after transfection, the cells were treated with rhodamine-conjugated transferrin for 10 min, then fixed and stained with DAPI. DN-amph-1 GFP (GFP fused with dominant negative mutant of amphiphysin-1 in transfected cells, a); internalized rhodamine-conjugated transferrin (b) and DAPI (c) were visualized by a confocal microscope. Bar, 10 µm. B) Transferrin uptake was quantitatively measured by counting rhodamine-positive cells from GFP-negative and -positive cells. The data are representative of four separate experiments. Bars represent the range of triplicate determinations.

4. Microinjection of amphiphysin-1 gene could restore the receptor-mediated endocytotic function of senescent fibroblasts
Senescent fibroblasts were microinjected with amphiphysin-1 cDNA under the regulation of CMV promoter. The expression of amphiphysin-1 was detected in the injected cells by immunofluorescent staining against amphiphysin-1 as well as coinjected markers such as rabbit IgG (Fig. 2A ). Those wild-type amphiphysin-1-reconstituted senescent cells were challenged by fluorescence-conjugated transferrin to check the clathrin-dependent, receptor-mediated endocytosis activity. As shown in Fig. 2B , endocytotic activity of senescent cells was sharply increased by the introduction of amphiphysin-1 cDNA. These results suggest that amphiphysin-1 is sufficient for the restoration of functional endocytosis of the senescent fibroblasts.

View larger version (15K): [in this window] [in a new window]   Figure 2. Restoration of endocytotic activity in senescent fibroblasts by microinjection of amphiphysin-1 cDNA. A) Senescent fibroblasts were microinjected with pcDNA 3.1 or amphiphysin-1 cDNA cloned in pcDNA3.1 with rabbit IgG protein as an injection control. In a separate set of experiments, coinjected rabbit IgG was stained with rhodamine (a) or FITC-conjugated (c) anti-rabbit IgG at 24 h after injection. Expression of amphiphysin-1 in injected cell was stained with monoclonal anti-amphiphysin-1 antibody and FITC-conjugated anti-mouse IgG antibody (b). Microinjected cells were loaded with rhodamine-conjugated transferrin for 10 min and fixed cells were morphologically analyzed by a fluorescence microscope (d). B) Transferrin uptake was quantitatively measured by counting rhodamine-positive cells of FITC-positive cells. Each bar represents the means of the triplicate experiments.

CONCLUSIONS AND SIGNIFICANCE
In the present study, we have attempted to monitor the functional capacity and molecular mechanism for the clathrin-dependent receptor-mediated endocytosis in the senescent cells. Unlike their presenescent counterparts, senescent HDFs showed reduced uptake of transferrin, suggesting that efficacy of receptor-mediated endocytosis in the senescent cells was significantly deteriorated. As a possible mechanism for the reduced activity of receptor-mediated endocytosis, we clearly showed the decrease in amphiphysin-1 protein expression not only in the multipassaged senescent human foreskin diploid fibroblasts and IMR 90 cells (fetal lung fibroblasts), but also in the hydrogen peroxide-induced premature senescent cells. The functional significance of amphiphysin-1 in the receptor-mediated endocytosis was confirmed by the transfection of dominant negative mutant of amphiphysin-1 into presenescent fibroblasts, resulting in the loss of transferrin uptake (Fig. 1A , B ). Moreover, we could revert the endocytotic activity of the senescent cells by illustrating the active transferrin uptake through simple microinjection of wild-type amphiphysin-1 into those cells (Fig. 2A , B ). These results suggest that the amphiphysin-1 plays a critical role in the reduction of receptor-mediated endocytosis of the senescent fibroblasts.

The process of receptor-mediated endocytosis is composed of several steps, which include recruitment of the clathrin coats and fission of the coated bud. After the receptor conjugation by ligand, such as EGF, receptor tyrosine kinase phosphorylates clathrin, which can provide a binding site for amphiphysin. Amphiphysin-1 is suggested to be involved in the recruitment and oligomerization at the neck of endocytotic buds. Amphiphysin-1 bridges the AP-2/clathrin coat and dynamin-1 to make an endosomal vesicle. The carboxyl-terminal domain of amphiphysin recruits GTPase dynamin to pinch off the coated buds. Amphiphysin acts as a regulated linker protein that couples clathrin-mediated budding of endocytotic vesicles to dynamin-mediated vesicle fission. We have shown here that the sole alteration of amphiphysin-1 function could completely inhibit the transferrin uptake. Moreover, it is interesting that of all the proteins associated with receptor-mediated endocytosis studied in the present work, only amphiphysin-1 is reduced in the senescent cells. The other proteins involved in receptor-mediated endocytosis, such as transferrin receptor, clathrin, dynamin, -adaptin, and ß-adaptin, were not changed in their expressions in senescent HDFs. These data strongly implicate the biological significance of amphiphysin-1 in functional deterioration of the senescent cells, though the mechanism for the down-regulation of amphiphysin-1 in the senescent cells requires a further study (Fig. 3 ).

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic diagram of the possible molecular mechanism of the decrement of endocytotic activity in senescent cells.

The functional recovery of the transferrin uptake in senescent cells by simple microinjection of amphiphysin-1 gene suggests the possibility of resuming receptor-mediated endocytotic activity of the senescent cells. With the present data, the role of amphiphysin-1 in receptor-mediated endocytosis of the senescent cells has been clearly analyzed and the possibility of the application of amphiphysin-1 for the functional recovery of the senescent cells, especially in relation to receptor-mediated endocytosis, has been suggested.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0723fje ; to cite this article, use FASEB J. (May 9, 2001) 10.1096/fj.00-0723fje

² These two authors are equal contributors to this work.

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This Article Full Text (PDF) Erratum All Versions of this Article: 15/9/1625 00-0723fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by PARK, J.-S. Articles by PARK, S. C. Search for Related Content PubMed PubMed Citation Articles by PARK, J.-S. Articles by PARK, S. C.