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1. Introduction

Patients with Werner syndrome (WS) show a wide range of premature aging phenotypes accompanied by rare tumors (1). The major premature aging phenotypes include gray hair, hoarseness, cataract, diabetes and malignancy (Table I). However, phenotypes of WS are not necessarily identical to those of natural aging; for instance WS is not usually accompanied by hearing loss, presbyopia or brain dysfunction, such as Alzheimer’s disease and Parkinson’s disease (1,2).

Table I Major phenotypes of Werner syndrome.

Table I

Major phenotypes of Werner syndrome.

Phenotypes	Occurrence frequency (%)	Mean age (years)	Oxidative stress and inflammation	Suppressed cell growth
Growth retardation	100	18.9	-	Yes
Gray hair, alopecia	100	20.1	Yes (Gray hair)	Yes (alopecia)
Hoarseness	100	22.8	Yes	Yes
Skin sclerosis	100	26.4	Yes	-
Cataract	100	31.2	Yes	-
Diabetes mellitus	70	31.5	Yes	-
Skin ulcer	40	34.7	Yes	Yes
Hypogonadism	80	35.6	Yes	Yes
Osteoporosis	60	39.5	Yes	Yes
Immune abnormalities	80	40.0	Yes	Yes
Atherosclerosis	20	40.6	Yes	-
Brain atrophy	40	40.7	Yes	Yes
Malignancy	20	41.3	Yes	-

[i] This table is based on reference (1). Yes, known or suggested; -, unknown.

WRN, the causative gene of WS, is located on human chromosome 8p12-11.2 (3). This gene encodes the WRN protein consisting of 1432 amino acids (4), which acts as a DNA helicase with exonuclease activity (5–7). WRN is a member of the RecQ helicase gene family, which includes RECQL1 (8), Bloom syndrome gene (BLM) (9), WRN (4), Rothmund-Thomson syndrome gene (RTS/RECQL4) (10), and RECQL5 (11). Mutations of BLM, WRN and RTS result in Bloom syndrome (BS), Werner syndrome (WS) and Rothmund-Thomson syndrome (RTS), respectively. These three genetic disorders are associated with genomic instability, and therefore the RecQ helicases are considered to be guardians of the genome (12–14). Diseases caused by mutations of RECQL1 and RECQL5 have yet to be identified.

WS has ~100 different mutation types, which result in the early termination of protein synthesis (15). The WRN protein has a nuclear localization signal (NLS) in its C-terminus and is located in the nucleus where it functions. Mutations occur throughout the helicase molecule resulting in truncated polypeptides that lack NLS in the C-terminus (16). Therefore, a mutated WRN in patient cells cannot be transported to the nucleus (17,18).

The mechanism of how the mutation of the single gene WRN induces such multi-phenotypes in WS remains to be determined. This perspective review focused on a hypothetical cascade beginning with telomere dysfunction caused by WRN gene mutation leading to multi-phenotypes of premature aging and an abnormal profile of tumors.

2. Genomic instability and telomere dysfunction

WS somatic cells are characterized by chromosomal aberrations known as ‘variegated translocation mosaicism’ (19), representing frequent pseudodiploidy with variable and clonal structural rearrangements associated with a high proportion of genomic deletions (20,21). This type of chromosomal aberration is assumed to be closely correlated with premature aging phenotypes, including rare tumors, in WS.

Accelerated loss of telomere repeats was observed in cultured WS patient fibroblasts (22), and recently this phenomenon was also confirmed in vivo by Ishikawa et al (23), who noted an accelerated epidermal telomere loss in WS patient tissues (Fig. 1). Authors of that study analyzed statistically the relationship between WS and control groups by applying a multiple regression model to the data of Fig. 1, and concluded that the lengths of the terminal restriction fragment (TRF) in WS patients were equivalent to those in control individuals who were ≥26 years. Abnormal telomere changes of B-lymphoblastoid cell lines (LCLs) from WS patients transformed by Epstein-Barr virus have also been previously reported (24) (Fig. 2). The telomere length of the majority of LCLs from normal individuals decreases uniformly and most of them no longer proliferate by 160 population doublings, although a very small proportion of them immortalize to continue proliferation (24–27). However, most LCLs obtained from WS patients show irregular changes of telomere length and repeated lengthening and shortening, without the occurrence of immortalized cell lines. We hypothesized (28) that an alternative pathway is involved in maintaining telomeres (29) in order to maintain telomere length in normal and WS LCLs that have relatively long life spans (28) compared with normal fibroblasts (30).

Figure 1 Terminal restriction fragment (TRF) length in skin samples from Werner syndrome (WS) patients and controls. This figure is based on reference (23). ●, WS patients; ○, controls.

Figure 2 Abnormal telomere changes in B-lymphoblastoid cell lines (LCLs) of Werner syndrome (WS). This figure is based on the figure from reference (28). Cell line WS11301 from a WS patient (dotted line) is mortal LCL with a lifespan of 150 PDL, showing irregular changes of the telomere. The cell line N0003 from a normal individual (solid line) is an immortalized LCL that decreased the telomere length uniformly to 135 PDL and, thereafter is immortalized by expressing high telomerase activity and maintained telomere length.

Molecular biological studies support the idea that genomic instability in WS cells is caused directly by telomere dysfunction. WRN helicase is capable of unwinding in vitro duplexed and tetraplexed DNA structures occurring in telomere and ribosomal DNA that are rich in repeated sequences (31). WRN is considered necessary for the efficient replication of G-rich telomere DNA, preventing telomere dysfunction and consequent genomic instability (32). Replication-associated telomere loss is responsible for chromosome fusions found in WS fibroblasts (33).

When the supF shuttle vector (SV) with (TTAGGG)6 as a model of a telomere was used for the mutagenesis assay in order to evaluate the role of WRN protein (34), SV sequences were stably replicated in human cells having normal WRN protein. WRN depletion, however, caused a marked increase (70-fold) in deletions and rearrangements arising within telomere SV. These results suggest that WRN protein contributes to the prevention of large deletions and rearrangements during replication of a telomere sequence, and provides a possible explanation for increased telomere loss and abnormal telomere dynamics in WS cells.

The shelterin complex is formed at telomeres by a group of six proteins including telomeric repeat binding factors TRF1 and TRF2, which assemble along the telomere region and are involved in telomere maintenance and protection (35–37). Of note, WRN is assumed to operate preferentially on aberrant DNA structures believed to exist in vivo, such as replication of forked DNA, Holliday junctions, triplex and tetraplex DNA, and to repair partial duplex with single-stranded bubble (38). WRN has also been shown to cooperate functionally at telomeres with shelterin proteins, including TRF2 (38). Therefore, this evidence strengthens the idea that absence of normal WRN in WS cells may be closely associated with the telomere dysfunction of WS cells.

Telomere dysfunction by an extreme shortening requires a long period of time and therefore, the present hypothesis regarding involvement of telomere dysfunction in WS disorder explains the relatively late onset of its phenotypes (1), at approximately age 20.

3. Telomere dysfunction and activation of aging pathway

WS cells prolong S-phase and other abnormalities in DNA replication (39–41). DNA damage, including telomere dysfunction, arrests cell growth leading to aging (42), and a hypothesis was proposed for natural aging where telomere-based aging is primarily a stem cell defect caused by the activation of p53 and then by the induction of growth arrest, senescence and apoptosis in resident stem and progenitor cells (43). Davis et al (44) investigated the signaling pathways involved in the proliferative life span barriers in WS fibroblasts. Cultured WS fibroblasts undergo senescence after ~20 population doublings, which was associated with high levels of CdkIs p16 and p21. Of note, senescent WS cells reentered the cell cycle following microinjection of a p53-neutralizing antibody. Davis et al (44) concluded that the strong similarity between signaling pathways triggering cell cycle arrest in WS and normal fibroblasts supports the hypothesis that accelerated loss of telomeres in WS cells also leads to acceleration of a pathway of aging similar to that in normal cells. Therefore, some phenotypes of WS (Table I) may be induced by p53 activation, including growth retardation, alopecia, and hypogonadism, as they all seem to be correlated with cell proliferation.

An essential role of limiting telomeres in the pathogenesis of WS is also supported by experiments using late-generation mice that were null for WRN and TERC (telomerase RNA component) (45): these TERC-null mice have extremely shortened telomeres. These mice manifest phenotypes of WS patients, including premature aging phenotypes and unique tumors.

4. Mitochondrial compromise by telomere dysfunction

Additional mechanisms of aging by telomere dysfunction have been suggested. Recent studies using telomerase reverse transcriptase (TERT)-deficient mice with telomere dysfunction showed a marked compromised mitochondrial function (42,46). This mitochondrial change seems to be caused by combined suppression of transcriptional co-activators PGC1α (proliferator-activated receptor-γ coactivator-1α) and PGC1β and their downstream targets. This suppression was mediated by the direct binding of p53 to the promoters of PGC1α and PGC1β. Notably, TERT-deficient mice showed a reduced expression of genes essential for gluconeogenesis, β-oxidation and defense against reactive oxygen species (ROS) suggesting a mitochondrial compromise. A hypothesis of a telomere-mitochondrion connection was indicated from these results (42) that assumes that compromised mitochondrial function causes oxidative stress by increasing ROS levels leading to inflammation and various aging phenotypes. The WRN protein suppresses hypoxia-inducible factor-1 (HIF-1) complex (47). HIF-1 activation in WS cells in the absence of WRN participates in the generation of mitochondrial ROS, Therefore, mitochondrial ROS is considered to be activated in WS cells by a mechanism that includes HIF-1 participation.

Recently, two distinct teams provided evidence that Rothmund-Thomson syndrome (RECQL4) helicases play a role in mitochondrial DNA integrity, which is strongly involved in the aging process (48,49). The role of WRN in mitochondrial function, however, remains to be determined.

5. Increase in oxidative stress in WS

Pagano et al (50) suggested participation of oxidative stress in causing WS multi-phenotypes. For instance, they showed an in vivo prooxidant state in WS (51). In this correlation, Goto et al (52) showed that the disulfide glutathione:glutathione ratio was significantly altered in WS patients, glyoxal and methylglyoxal levels significantly increased, and the plasma levels of uric acid (52,53) increased significantly in WS patients. Vitamin C restored healthy aging phenotypes in a mouse model for Werner syndrome (54).

A significant contribution to human aging of low-grade, chronic and systemic inflammation caused by an imbalance between pro- and anti-inflammatory circuits, mainly by monitoring highly sensitive C-reactive protein (hsCRP), has recently been proposed as inflammaging to explain the aging mechanism (55). Goto et al (56) investigated the inflammatory condition associated with normal human aging by examining hsCRP in sera collected from healthy Japanese individuals and mutation-proven Japanese WS patients. The serum hsCRP level increased significantly with normal aging in males and females significantly increased in WS compared with age-matched normal and normal elderly populations. Accordingly, both normal aging and WS were associated with minor inflammation that can be evaluated by serum hsCRP.

Notably, cataract and type-II diabetes mellitus are associated with ROS in non-WS individuals and therefore these two phenotypes of WS may also be associated with ROS. Other phenotypes (Table I), such as gray hair, cataract, diabetes mellitus, skin ulcer and atherosclerosis, of WS may also correlate with ROS (50,57).

Correlated with the association of ROS, oxidative stress markers, including pentosidine and homocysteine, were examined in serum from WS patients and healthy individuals (58). Increased serum pentosidine correlated significantly with normal aging in healthy individuals. Serum pentosidine in WS patients increased significantly compared with age-matched healthy individuals. Serum homocysteine levels increased significantly with normal aging in healthy individuals, but those in WS patients did not increase compared with those from age-matched healthy individuals.

Linkage of increased inflammation and ROS in WS with mitochondrial compromise caused by telomere dysfunction seems likely. Fig. 3 summarizes a hypothetical cascade starting with WRN gene mutation and leading to multi-phenotypes of premature aging in WS patients. A body of data examining the effect of oxidative stress on inflammation by means of modified lipid metabolism has accumulated: for instance, oxidative stress is suggested to cause atherosclerosis and cardiovascular disease by the formation of pro-inflammatory, pro-atherogenic oxidized low-density lipoprotein (59).

Figure 3 A cascade of Werner syndrome (WS) pathogenesis starting with WRN gene mutation and leading to multi-phenotypes of premature aging and abnormality in tumorigenesis. This scheme does not cover all possible mechanism of the WS pathogenesis but focuses only on the WRN-telomere axis.

WRN plays a role in various functions not mediated by telomeres, such as repair of damaged DNA by genotoxins including camptothecin (60–62) and in the transcription of ribosomal RNA (63). These functions may also be relevant to the premature aging phenotypes of WS. Thus, Fig. 3 shows a major route focusing on the cascade beginning with the WRN-telomere axis.

6. Correlation with the target of rapamycin (TOR)

The target of rapamycin (TOR) is a conserved Ser/Thr kinase that regulates cell growth and metabolism in response to environmental cues (64). Inhibition of TOR extends the lifespan of invertebrates as well as of mammals (65–67). In a study conducted to determine whether stressed cells undergo cell death, reversible quiescence or irreversible senescence, p53 was shown to communicate with the mammalian TOR (mTOR), thereby adding yet another level of complexity to the signaling network that emanates from p53 (68). Talaei et al (69) found an increase in cytosolic aggregates in cultured WS fibroblasts and hypothesized that the phenotype is indirectly related to excess activation of the mTOR pathway, leading to the formation of protein aggregates in the cytosol with increasing levels of oxidative stress. As those authors found that the expression levels of the two main H2S-producing enzymes, cystathionine β synthase and cystathionine γ lyase, were lower in WS cells compared with normal cells, they investigated the effect of the administration of H2S by using NaHS (50 μM). NaHS treatment blocked mTOR activity, abrogated protein aggregation and normalized the phenotype of WS cells. Similar results were obtained by treatment with mTOR inhibitor rapamycin. These findings suggest the participation of mTOR in the pathogenesis of WS, although p53 activation with the mTOR system remains to be clarified.

7. Abnormality in tumorigenesis

Another characteristic feature of WS is a much higher incidence of rare tumors (70). Non-epithelial tumors, including soft-tissue sarcoma and benign meningioma, are highly associated with WS. Notably, the ratio of epithelial (55 cases) to non-epithelial tumors (76 cases) is 1:1.38 in WS patients (Table II) (71) compared with 10:1 in the general population (70,71). Genomic instability and chromosomal aberrations in WS may be the basic phenomenon leading to tumorigenesis, as hypothesized by Monnat (72). WRN helicase was shown to be required for immortalization accompanied by activation of the hTERT gene to activate telomerase by way of the telomere crisis pathway (TCP) in a system that uses LCLs transformed by the Epstein-Barr virus (62,73) (Table III). These data support the hypothesis that the development of TCP-mediated epithelial tumors also requires WRN helicase accompanied by telomerase activation. In non-WS individuals, telomere crisis is considered to produce significant chromosomal instability and thus is a hallmark of human cancer (reviewed in refs. 62,74), such as renal cell carcinoma (75). Whether the assumed inability of tumorigenesis by way of TCP due to WRN dysfunction is associated with an abnormal tumor profile in WS remains to be clarified.

Table II Neoplasms in Japanese Werner syndrome (1996–2008).

Table II

Neoplasms in Japanese Werner syndrome (1996–2008).

Diagnosis	No.
Non-epithelial
Soft-tissue sarcoma
MFH	8
Others	12
Osteosarcoma	6
Malignant melanoma	18
Meningioma	9
Hematologic disorders
AML	4
MDS	11
Others	8
Epithelial
Thyroid	9
Liver	6
Skin	5
Lung	5
Others	30
Total	131

[i] MFH, malignant fibrous histiocytoma; AML, acute myelogenous leukemia; MDS, myelodysplastic syndrome. This table is based on reference (71).

Table III Incidence of immortalization by way of the TCP of LCLs from non-WS and WS individuals.

Table III

Incidence of immortalization by way of the TCP of LCLs from non-WS and WS individuals.

Variables	Total LCLs	Immortalized	% of immortalization
Non-WS	61	10	16.4
WS	44	0	0

[i] This table is based on reference (73). Non-WS LCL samples include 11 LCLs from diabetes families showing a high incidence of immortalization (5/11) as well as remaining 50 normal LCLs (5/50). The Chi-square test of WS LCLs (0/44) against normal individuals (0/50) and the diabetes families (5/11) showed significant differences, P<0.031 and P<0.00001, respectively. TCP, telomere crisis pathway; LCLs, B-lymphoblastoid cell lines; WS, Werner syndrome.

A hypothetical scheme explaining the role of WRN helicase in immortalization by a supposed ‘breakage-fusion-bridge cycle’ of chromosomes at telomere crisis (76) was suggested in correlation with the unique tumorigenesis profile in WS (77). WRN helicase may have at least two mutually compatible roles in immortalization by way of TCP. First, WRN helicase may unwind the repressed state of chromatin DNA, leading to modification and activation of the promoter region of the hTERT gene (78). Second, in the telomerase-mediated de novo addition of telomeres to non-telomeric sequences generated during the ‘breakage-fusion-bridge cycle’ (76), the exonuclease activity of WRN helicase may also be involved in this process to trim the 3′ end to expose a favorable sequence as a primer for adding a telomere to the non-telomeric end (79). At this point, particular telomeric repeats may be added by telomerase onto the 3′ end of non-telomeric primers. WRN has been shown to cooperate functionally at telomeres with shelterin proteins, including TRF2 (37), which supports the hypothetical function of WRN in immortalization by way of TCP, as previously suggested (77).

8. Conclusion

This perspective review has shown that the majority of wide and complex premature aging phenotypes, including abnormal tumor profiles, of WS may be explained in a unified manner by the cascade beginning with telomere dysfunction initiated by WRN gene mutation, leading to mitochondrial dysfunction and overproduction of ROS.

Acknowledgements

We would like to thank Dr Yasuhiro Furuichi of the GeneCare Research Institute, Co., Ltd. for his invaluable discussion in the preparation of this manuscript. We would also like to thank Dr Kaiyo Takubo, the Tokyo Metropolitan Institute of Gerontology, for his important input.

References