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. 2004 Nov 26;64(6):816–823. doi: 10.1136/ard.2004.025445

Increased expression of humanin peptide in diffuse-type pigmented villonodular synovitis: implication of its mitochondrial abnormality

K Ijiri 1, H Tsuruga 1, H Sakakima 1, K Tomita 1, N Taniguchi 1, K Shimoonoda 1, S Komiya 1, M Goldring 1, H Majima 1, T Matsuyama 1
PMCID: PMC1755539  PMID: 15567815

Abstract

Objectives: To define the pathogenesis of pigmented villonodular synovitis (PVNS), by searching for highly expressed genes in primary synovial cells from patients with PVNS.

Methods: A combination of subtraction cloning and Southern colony hybridisation was used to detect highly expressed genes in PVNS in comparison with rheumatoid synovial cells. Northern hybridisation was performed to confirm the differential expression of the humanin gene in PVNS. Expression of the humanin peptide was analysed by western blotting and immunohistochemistry. Electron microscopic immunohistochemistry was performed to investigate the distribution of this peptide within the cell.

Results: 68 highly expressed genes were identified in PVNS. Humanin genes were strongly expressed in diffuse-type PVNS, but were barely detected in nodular-type PVNS, rheumatoid arthritis, or osteoarthritis. Humanin peptide was identified in synovium from diffuse-type PVNS, and most of the positive cells were distributed in the deep layer of the synovial tissue. Double staining with anti-humanin and anti-heat shock protein 60 showed that humanin was expressed mainly in mitochondria. Electron microscopy disclosed immunolocalisation of this peptide, predominantly around dense iron deposits within the siderosome.

Conclusions: Increased expression of the humanin peptide in mitochondria and siderosomes is characteristic of synovial cells from diffuse-type PVNS. Humanin is an anti-apoptotic peptide which is encoded in the mitochondrial genome. Present findings suggest that mitochondrial dysfunction may be the principal factor in pathogenesis of diffuse-type PVNS and that humanin peptide may play a part in the neoplastic process in this form of PVNS.

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Figure 2.

Figure 2

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 Northern blot analysis of mRNAs expressed by synovial cells from patients with PVNS, RA, and OA. Total RNA (168 ng) was subjected to electrophoresis in a 1.0% agarose gel containing formaldehyde, transferred to a nylon membrane, and probed with [32P]dCTP labelled cDNA (type 9; fig 1). Another cDNA (type 3) encoded in the 16S rRNA region was also used in northern blotting and the expression level and size were same as those using type 9 cDNA (data not shown). Humanin genes were strongly expressed in diffuse-type PVNS, but barely detected in nodular-type PVNS, RA, or OA. The size of the expressed major message was ∼1.6 kb and the other messages were ∼1 kb, which corresponds to the results of a previous report by Hashimoto et al.28

Figure 1.

Figure 1

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 The sequences encoded within the 16S rRNA region with poly A tail. The cDNA fragments were aligned with the 16S rRNA region of the mitochondrial gene and the correlating humanin mRNA sequence. Southern colony hybridisations repeated these sequences in a total of three rounds independently. Oblique bars show the digestion sites by Rsa I and upward diagonal bar shows the region of humanin coding sequences. Although there are nine types of sequences with poly A tail within this region, only the type 9 sequence was identical to the previously reported mRNA encoding humanin peptide.

Figure 3.

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 The expression of genes encoded in mitochondria other than humanin genes. Total RNA was extracted from the synovial cells of five patients with PVNS, three with RA, and three with OA, and NADH dehydrogenase, ATPase 6, cytochrome c, cytochrome b, and GAPDH mRNA levels were analysed by semiquantitative RT-PCR. The levels of expression of these genes in PVNS were not increased in other types of arthritis, indicating that the humanin gene was selectively expressed in mitochondrial genes in PVNS.

Figure 4.

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 Expression of humanin peptide in synovial cells from diffuse-type PVNS. Protein (20 µg) from synovial cell lysates was subjected to SDS-PAGE on a 5–20% gradient gel. Rabbit anti-humanin polyclonal antibody was used for western blotting. Synthesised peptide, which was used as antigen to produce rabbit anti-humanin polyclonal antibody, was used as a standard and rabbit IgG was used as a negative control.

Figure 5.

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 Synovial tissue from diffuse-type PVNS was fixed with 4% formaldehyde in PBS. The specimens were stained with anti-humanin antibody, followed by Alexa 488 goat antirabbit IgG, and photographed with a fluorescent microscope (x40). (A) Most positive cells (green) were distributed in a deep layer with haemosiderin deposit. (C) Negative control of the continuous section. (B, D) Backgrounds for (A) or (C), respectively.

Figure 6.

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 Relationship between humanin peptide expression and mitochondria. Isolated synovial cells containing haemosiderin were double stained with anti-humanin antibody and anti hsp60 antibody as first antibodies, followed by goat antirabbit IgG and donkey antigoat IgG as second antibodies (x400). (A) Haemosiderin was deposited unequally throughout the cytoplasm. (B) Single anti-humanin antibody staining (red). (C) Single anti-hsp60 antibody staining (mitochondrial staining; green). (D) Humanin was dominantly distributed in the mitochondria around the siderosome (yellow).

Figure 7.

Figure 7

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 Electron micrograph of synovial cells from diffuse-type PVNS. Most of the electron dense iron deposits were observed within the siderosomes. Some electron dense iron deposits were observed within mitochondria (arrows). (A) Mitochondrial membrane debris with electron dense deposits was observed within the siderosome as an autophagosome (left arrow). (B) Some of the normal mitochondria (arrows) also were scattered throughout the cytoplasm. (Magnification x19 000.)

Figure 8.

Figure 8

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 Electron microscopic immunohistochemistry of synovial cells from diffuse-type PVNS. (A) In some of the siderosomes, particles of colloidal gold, were precipitated in the debris adjacent to electron dense iron. These results demonstrate that humanin peptide is present within the debris that is phagocytosed into the siderosome. (B) Negative control for immunohistochemistry. (Magnification x29 000.)

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