Klotho


Here is the next in what will likely be a long series of semi-regular review roundups — links, without extensive further comment, to the reviews I found most intriguing over the past few weeks months (I went on hiatus during the winter holidays). For the previous foray into the secondary literature, see here.

Alzheimer’s:

Apoptosis & cancer:

Calorie restriction:

Diabetes:

Klotho:

Sirtuins:

Stem cells:

Telomeres:

The soluble protein Klotho was initially widely believed to be an anti-aging factor: knockout mice live short lives and display symptoms of segmental progeria, and increasing klotho gene dosage protects the kidneys against age-related decline. Mechanistic studies suggested that loss-of-function mutations in klotho derepress the Wnt signaling pathway, resulting in premature cellular senescence.

More recently, however, the relationship between Klotho and bona fide aging has been called into question — specifically by the claim that klotho mutants are suffering from hypervitaminosis D. While this syndrome can phenocopy some aspects of aging (so the argument goes) it does not represent premature aging as such: the klotho mutant phenotype can be partially rescued by placing mice on a diet low in vitamin D, a regimen that does nothing to slow normal aging. This has led some to argue that Klotho should no longer be considered a pro-longevity factor.

The tennis match continues, most recently with a forceful volley from the pro-“klotho as anti-aging gene” side of the court. Wolf et al. remind us that Klotho downregulates the IGF-I signaling pathway (in which loss-of-function mutation generally extends lifespan; this is consistent with a bona fide anti-aging role for Klotho), and describe the gene’s effects on the proliferation of breast cancer cell lines:

Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer

Klotho is an anti-aging gene, which has been shown to inhibit the insulin and insulin-like growth factor 1 (IGF-1) pathways in mice hepatocytes and myocytes. As IGF-1 and insulin regulate proliferation, survival and metastasis of breast cancer, we studied klotho expression and activities in human breast cancer. Immunohistochemistry analysis of klotho expression in breast tissue arrays revealed high klotho expression in normal breast samples, but very low expression in breast cancer. In cancer samples, high klotho expression was associated with smaller tumor size and reduced KI67 staining. Forced expression of klotho reduced proliferation of MCF-7 and MDA-MB-231 breast cancer cells, whereas klotho silencing in MCF-7 cells, which normally express klotho, enhanced proliferation. Moreover, forced expression of klotho in these cells, or treatment with soluble klotho, inhibited the activation of IGF-1 and insulin pathways, and induced upregulation of the transcription factor CCAAT/enhancer-binding protein beta, a breast cancer growth inhibitor that is negatively regulated by the IGF-1-AKT axis. Co-immunoprecipitation revealed an interaction between klotho and the IGF-1 receptor. Klotho is also a known modulator of the fibroblast growth factor (FGF) pathway, a pathway that inhibits proliferation of breast cancer cells. Studies in breast cancer cells revealed increased activation of the FGF pathway by basic FGF following klotho overexpression. Klotho did not affect activation of the epidermal growth factor pathway in breast cancer cells. These data suggest klotho as a potential tumor suppressor and identify it as an inhibitor of the IGF-1 pathway and activator of the FGF pathway in human breast cancer.

In summary, then: As in other systems, Klotho downregulates the IGF-1 pathway in breast cancer (from the co-IPs, it looks like it might be acting as a receptor antagonist). Systemic downregulation of the IGF-1 pathway results in lifespan extension; here, it appears to slow proliferation of two cultured breast cancer cell lines with rather different behavioral profiles (MB-231 is quite aggressive, whereas MCF-7 is more genteel). From the tissue arrays, the Klotho expression pattern behaves as one would expect for a tumor suppressor: high in normal breast tissue but low in tumor cells; within tumors, higher Klotho correlates with endpoints associated with better outcomes (smaller tumors and lower levels of proliferation markers).

Within the larger context of the Klotho controversy, this study is a staunch argument in favor of continued study of the gene as an anti-tumor and anti-aging factor: in this system at least, Klotho is slowing tumor growth and inactivating a gerontogene. ( Let us take as read the somewhat bitchy criticism that most of the main results are from cell culture, and that it would be nice to see confirmation in whole-organism studies.) Thus, the results deal a strong blow against the claim that the Klotho phenotypes are an epiphenomenon resulting from a derangement of vitamin metabolism. After all, one can’t cure cancer by withholding vitamin D.

The soluble protein Klotho appears to be an anti-aging factor, since mice deficient in the Klotho gene show signs of premature aging. However, the validity of Klotho-/- mutants as a model of progeria is controversial: many of the pathological features of the mutant phenotype can be attributed to hypervitaminosis D, and can be reversed by eliminating vitamin D from the diet. (Biogerontologists are generally more skeptical of progeria than increased longevity, since there are lots of ways to shorten lifespan that don’t involve bona fide accelerating of the aging process, whereas there are far fewer ways to lengthen lifespan without slowing that process down.)

Another blow against the idea of Klotho as a regulator of lifespan comes from Brownstein et al., who show that increased circulating levels of Klotho protein (pursuant to a chromosomal translocation that activates the gene) are associated with hyperparathyroidism and rickets (n.b. that the latter is a classic symptom of, wait for it, vitamin D deficiency).

This is interesting from an endocrinological standpoint because rickets is often the result of hypophosphatemia (low phosphate –> weak bones), whereas hyperparathyroidism is usually associated with hyperphosphatemia.

But from a biogerontological perspective, it’s both interesting and sad: Specifically, it’s a strike against the idea that we might be able to supplement aging mammals (like ourselves) with increased levels of Klotho in order to forestall aging. Of course, it’s possible that the very elevated levels of the protein seen in this study are totally off the charts, and that more modest doses might have a salubrious effect — but more and more, the most reasonable interpretation of the data is that Klotho is involved in mineral and vitamin metabolism in a way that doesn’t have much to do with lifespan; that the levels of Klotho are already more or less optimized (since a syndrome results from either deficiency or elevation relative to wildtype); and that the “progeria” of Klotho-/- is simply a compelling mimic of accelerated aging rather than a legitimate model of the process.

The Wnt signaling pathway has been implicated in accelerating the aging process by interfering with the action of Klotho, but this claim has met with resistance; there is also emerging evidence that some of Wnt’s effects delay age-related decline in specific tissues. A recent review by DeCarolis et al. describes the controversy and weighs the arguments on all sides.

Another piece by Makato Kuro-o, focused primarily on mechanisms of Klotho action, also discusses the relationship between Klotho and Wnt.

Earlier this year, we discussed a pair of papers that proposed a role for the Wnt signaling pathway in aging. One of those studies focused on the klotho-/- mouse, which shows signs of progeria and has been taken as a model of accelerated aging.

In a letter to Science last week, however, we’re given cause to reconsider these results: U-Michigan’s Richard Miller, one of the more erudite and thoughtful (and outspoken) eminences grises of biogerontology, reminds us of some recent findings might influence our interpretation of any experiment using the klotho-/- mutant:

Liu et al. (Report, “Augmented Wnt signaling in a mammalian model of accelerated aging,” 10 August, p. 803) have elegantly shown how alterations in Wnt signals contribute to the suffering of klotho-deficient mice, but not every sick little rodent is a suitable model for human aging. The pathological features and short life span of klotho mutant mice have been shown to reflect hypervitaminosis D, secondary to ablated responses to Fgf-23 (1-3). The same syndrome appears in Fgf-23 mutants and can be cured by deleting the 1–hydroxylase gene that increases the activity of the vitamin. In both mutants, the features represented as evidence of “premature aging” can be eliminated simply by putting the mice on a diet low in vitamin D. Perhaps vitamin D deprivation will turn out to be the long-sought cure for aging, but in the meantime, it would be wise to view with some skepticism the claims that klotho and similar developmental mishaps provide convenient shortcuts for learning about mechanisms of “real” aging.

In other words, if the klotho-/- phenotype can be cured by twiddling the dietary levels of a single essential vitamin, to what extent can it be considered a legitimate model of accelerated aging? By extension, to what extent does this force a re-evaluation of findings based on the idea that klotho-/- is a bona fide progeria model?

Two authors of the study in question, Hongjun Liu and PI Toren Finkel, respond in the same forum, as is their right:

There are many areas in aging research in which there is some disagreement. One question in dispute is the degree to which observations in simple organisms, such as postmitotic worms, can inform our understanding of mammalian aging. Similarly, reasonable people disagree on the role, if any, of cellular senescence in organismal aging. We appreciate that there is also considerable disagreement regarding how much mammalian models of accelerated aging can teach us about the normal aging process.

Our study centered on a set of observations suggesting that the Wnt family of proteins could bind to klotho, a protein whose absence has been linked to an accelerated aging phenotype in mice. Genetic evidence suggests that alleles of klotho are also associated with variation in human longevity (1). Nonetheless, we agree with Miller that considerable care must be taken when using the existing accelerated aging models as an indication of the normal aging process. Our opinion is that studying models of rapid aging will be useful in teasing out the underlying mechanisms of how we age, although we understand that Miller does not share that opinion. Hopefully, we will all live long enough to find out who is right.

I think the answer is a bit thin, as it doesn’t address Miller’s fundamental objection to the model (that the klotho-/- phenotype has a fundamental difference from real aging, namely, its ability to be cured by vitamin D deprivation), but others may disagree. Obviously, as the old refrain goes, more research is necessary. In the meantime, however, I wouldn’t start taking any Wnt inhibitors just yet.

The Wnt signaling pathway, originally discovered in a developmental context, is now known to play a key role in the homeostasis of many tissues; furthermore, its signaling via ß–catenin (one of Wnt’s several receptors) is perturbed in a variety of tumors. Now two complementary papers have demonstrated that Wnt may play a causative role in aging.

The first study focuses on the klotho mouse model, in which a loss-of-function mutation results in accelerated aging. Liu et al. demonstrate that in wildtype mice, Klotho protein binds Wnt in the serum and thereby antagonizes Wnt interactions with its receptors. In the absence of Klotho (i.e., in the klotho-/- mutants), Wnt has free reign and augmented activity. The authors further demonstrate that high Wnt signaling accelerates cellular senescence, a phenomenon increasingly implicated in age-related decline in tissue function. (The identification of the specific receptor involved is left as a future exercise.)

The deleterious consequences of a hyperactive Wnt axis are elaborated by Brack et al., who show that increased Wnt signaling (in this case mediated via the less well-studied receptor Frizzled) is associated with a lineage conversion in myogenic progenitors. As Wnt activity increases during aging, muscle cell progenitors switch from a myogenic (i.e., regenerative) mode to a fibrogenic (i.e., inflammatory) mode; this can be prevented with specific blocking antibodies. It is easy to that the resulting increased fibrogenesis, at the cost of regenerative capacity, could cause muscular weakness and sarcopenia in late life.

Happily, some attention is already being paid to pharmaceutical intervention in Wnt pathways, in the context of seeking antagonists that might be useful in the treatment of cancer (see here and here). These efforts currently focus on the consequences of signaling via ß-catenin, which may or may not be relevant to the findings reported above (in the first paper, events downstream of ß-catenin are used as readouts of Wnt activity, but it’s not clear whether ß-catenin is actually required for the increase in senescence; in the second paper, it’s clear that a distinct receptor, Frizzled, at least plays a significant role). Nonetheless, if drug designers aim high enough in the pathway (i.e., at soluble Wnt, where the Klotho interaction is) they might be able to hit two birds with one stone.

The gene klotho encodes a secreted soluble protein that has been implicated in regulation of lifespan. In rodents, mutation in the gene results in a premature aging syndrome; in humans, specific alleles of the gene appear to improve one’s chances of living an unusually long life.

Klotho is expressed primarily in the kidney. In PNAS this week, Haruna et al. explore the role of the protein in a model of kidney disease. The results indicate that increase klotho gene dosage significantly protects the kidney against degenerative damage:

Klotho, an antiaging gene with restricted organ distribution, is mainly expressed in the kidney tubules; the mutant mice have shortened life span, arteriosclerosis, anemia, and osteoporesis, features common to patients with chronic renal failure. Conceivably, the reduction of the Klotho gene expression may contribute to the development of kidney failure; alternatively, its overexpression may lead to the amelioration of renal injury in an ICR-derived glomerulonephritis (ICGN) mouse model with subtle immune complex-mediated disease. To address this issue, four different strains of mice were generated by cross-breeding: ICGN mice without the Klotho transgene (ICGN), ICGN mice with the Klotho transgene (ICGN/klTG), wild-type mice with the Klotho transgene (klTG), and wild-type mice without the Klotho transgene (control). At 40 weeks old, the survival rate was approximately 30% in ICGN mice, and approximately 70% in the ICGN/klTG group. This improvement was associated with dramatic improvement in renal functions, morphological lesions, and cytochrome c oxidase activity but a reduction in beta-galactosidase activity (a senescence-associated protein), mitochondrial DNA fragmentation, superoxide anion generation, lipid peroxidation, and Bax protein expression and apoptosis. Interestingly, improvement was seen in both the tubular and glomerular compartments of the kidney, although Klotho is exclusively confined to the tubules, suggesting that its gene product has a remarkable renoprotective effect by potentially serving as a circulating hormone while mitigating the mitochondrial oxidative stress.

The results raise the possibility that the otherwise healthy klotho mutant mice (in which the lifespan phenotype was first detected) are dying young because of kidney problems. Is this the case? If so, is it the loss of kidney function per se or problems arising from degenerative cell loss that shorten lifespan? It’s a tough question to answer, because Klotho is a circulating factor. Hence the ideal experiment (transplanting klotho+/+ kidneys into klotho-/- mutant mice to restore normal kidney function) would be complicated by the fact that Klotho secreted by the wildtype kidney could act upon the mutant’s kidneys (and other organs). One would like to have a way of restoring kidney function without introducing more Klotho into the bloodstream, and then asking whether the mutant mice still display a premature aging phenotype.

Mouse dialysis, anyone?

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