Mutsuko Ohnishi

LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer

The CpG island methylator phenotype (CIMP) with widespread promoter CpG island methylation is a phenotype in colorectal cancer, associated with microsatellite instability (MSI) and BRAF mutation. Genome‐wide hypomethylation may also play an important role in genomic instability. However, the relation between global DNA methylation level and methylation in individual CpG islands remains uncertain. Utilizing 869 population‐based colorectal cancers, we measured long interspersed nucleotide element‐1 (LINE‐1) methylation level by Pyrosequencing, which correlates with global DNA methylation level. We quantified DNA methylation in 8 CIMP‐specific promoters (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) by real‐time PCR (MethyLight technology). LINE‐1 methylation levels in tumors were approximately normally distributed (mean, 61.4%; median, 62.3%; standard deviation, 9.6%). Among the 869 tumors, 128 (15%) were classified as CIMP‐high (≥6/8 methylated promoters). The mean LINE‐1 methylation level was higher in CIMP‐high tumors (65.1%, p < 0.0001) than non‐CIMP‐high tumors (60.7%), and higher in MSI‐high tumors (64.7%, p < 0.0001) than non‐MSI‐high tumors (60.7%). When tumors were stratified by MSI/CIMP status, compared to non‐MSI‐high non‐CIMP‐high tumors (mean LINE‐1 methylation level, 60.4%), the mean LINE‐1 methylation level was higher in MSI‐high CIMP‐high (64.8%, p < 0.0001), MSI‐high non‐CIMP‐high (64.6%, p = 0.03) and non‐MSI‐high CIMP‐high tumors (66.1%, p = 0.0003). In addition, 18q loss of heterozygosity in non‐MSI‐high tumors was correlated with LINE‐1 hypomethylation (p = 0.004). In conclusion, both CIMP‐high and MSI‐high are inversely associated with LINE‐1 hypomethylation, suggesting that CIMP/MSI and genomic hypomethylation may represent different pathways to colorectal cancer. Our data also support a possible link between global hypomethylation and chromosomal instability.

In vivo genetic evidence for klotho-dependent, fibroblast growth factor 23 (Fgf23) -mediated regulation of systemic phosphate homeostasis

A major breakthrough in systemic phosphate homeostasis regulation was achieved by the demonstration of strikingly similar physical, morphological, and biochemical phenotypes of fibroblast growth factor 23 (Fgf23) and klotho ablated mice, which led to identification of klotho as an Fgf23 signaling cofactor. Here, we generated Fgf23 and klotho double‐knockout (Fgf23−/−/klotho−/−) mice to test the hypothesis whether Fgf23 has a klotho‐independent function. Fgf23−/−/klotho−/− mice are viable and have high serum phosphate levels, similar to Fgf23−/− and klotho−/− single‐knockout mice. In addition, the Fgf23−/−/ klotho−/− mice have increased renal expression of the sodium/phosphate cotransporter NaPi2a and of 1‐alpha‐hydroxylase concomitant with increased serum levels of 1,25‐dihydroxyvitamin‐D, as also observed in the Fgf23−/− and klotho mice. Moreover, Fgf23−/−/ klotho−/− mice show soft tissue and vascular calcification, severe muscle wasting, hypogonadism, pulmonary emphysema, distention of intestinal wall, and skin atrophy, all of which are also seen in Fgf23−/− and klotho−/− mice. Notably, injection of bioactive FGF23 protein into Fgf23−/−/klotho−/− and klotho−/− mice does not lower serum phosphate, whereas in wild‐type and Fgf23−/− mice, it reduces serum phosphate. Together, these results provide compelling evidence that Fgf23 does not have a klotho‐independent role in the regulation of systemic phosphate and vitamin D homeostasis.— Nakatani, T., Sarraj, B., Ohnishi, M., Densmore, M. J., Taguchi, T., Goetz, R., Mohammadi, M., Lanske, B., Razzaque, M. S. In vivo genetic evidence for klotho‐dependent, fibroblast growth factor 23 (Fgf23) ‐mediated regulation of systemic phosphate homeostasis. FASEB J. 23, 433–441 (2009)

Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging

Identifying factors that accelerate the aging process can provide important therapeutic targets for slowing down this process. Misregulation of phosphate homeostasis has been noted in various skeletal, cardiac, and renal diseases, but the exact role of phosphate toxicity in mammalian aging is not clearly defined. Phosphate is widely distributed in the body and is involved in cell signaling, energy metabolism, nucleic acid synthesis, and the maintenance of acid‐base balance by urinary buffering. In this study, we used an in vivo genetic approach to determine the role of phosphate toxicity in mammalian aging. Klothoknockout mice (klotho−/−) have a short life span and show numerous physical, biochemical, and morphological features consistent with premature aging, including kyphosis, uncoordinated movement, hypogonadism, infertility, severe skeletal muscle wasting, emphysema, and osteopenia, as well as generalized atrophy of the skin, intestine, thymus, and spleen. Molecular and biochemical analyses suggest that increased renal activity of sodium‐phosphate cotransporters (NaPi2a) leads to severe hyperphosphatemia in klotho−/− mice. Genetically reducing serum phosphate levels in klotho−/− mice by generating a NaPi2a and klotho double‐knockout (NaPi2a−/−/klotho−/−) strain resulted in amelioration of premature aging‐like features. The NaPi2a−/−/klotho−/− double‐knockout mice regained reproductive ability, recovered their body weight, reduced their organ atrophy, and suppressed ectopic calcifications, with the resulting effect being prolonged survival. More important, when hyperphosphatemia was induced in NaPi2a−/−/klotho−/− mice by feeding with a highphosphate diet, premature aging‐like features reappeared, clearly suggesting that phosphate toxicity is the main cause of premature aging in klotho−/− mice. The results of our dietary and genetic manipulation studies provide in vivo evidence for phosphate toxicity accelerating the aging process and suggest a novel role for phosphate in mammalian aging.—Ohnishi, M., Razzaque, M. S. Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. FASEB J. 24, 3562–3571 (2010). www.fasebj.org

Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1α-hydroxylase

Changes in the expression of klotho, a beta-glucuronidase, contribute to the development of features that resemble those of premature aging, as well as chronic renal failure. Klotho knockout mice have increased expression of the sodium/phosphate cotransporter (NaPi2a) and 1alpha-hydroxylase in their kidneys, along with increased serum levels of phosphate and 1,25-dihydroxyvitamin D. These changes are associated with widespread soft-tissue calcifications, generalized tissue atrophy, and a shorter lifespan in the knockout mice. To determine the role of the increased vitamin D activities in klotho knockout animals, we generated klotho and 1alpha-hydroxylase double-knockout mice. These double mutants regained body weight and developed hypophosphatemia with a complete elimination of the soft-tissue and vascular calcifications that were routinely found in klotho knockout mice. The markedly increased serum fibroblast growth factor 23 and the abnormally low serum parathyroid hormone levels, typical of klotho knockout mice, were significantly reversed in the double-knockout animals. These in vivo studies suggest that vitamin D has a pathologic role in regulating abnormal mineral ion metabolism and soft-tissue anomalies of klotho-deficient mice.

Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model

Hyp mice possess a mutation that inactivates the phosphate‐regulating gene, which is ho‐mologous to the endopeptidases of the X‐chromo‐some (PHEX). The mutation is associated with severe hypophosphatemia due to excessive urinary phosphate wasting. Such urinary phosphate wasting in Hyp mice is associated with an increased serum accumulation of fibroblast growth factor (FGF) 23. We wanted to determine the biological significance of increased serum FGF23 levels and concomitant hy‐pophosphatemia in Hyp mice and to evaluate whether FGF23 activity could be modified by manipulating klotho (a cofactor of FGF23 signaling). We generated Hyp and klotho double‐mutant mice (Hyp/klotho−/−). Severe hypophosphatemia of Hyp mice was reversed to hyperphosphatemia in Hyp/klotho−/− double mutants, despite the fact that the double mutants showed significantly increased serum levels of FGF23. Hyperphosphatemia in Hyp/klotho−/− mice was associated with increased renal expression of sodium/phosphate cotransporter 2a (NaPi2a) protein. Exogenous injection of bioactive parathyroid hormone 1‐34 down‐regulated renal expression of NaPi2a and consequently reduced serum levels of phosphate in Hyp/klotho−/− mice. Moreover, in con‐trast to the Hyp mice, the Hyp/klotho−/− mice showed significantly higher serum levels of 1,25‐dihydroxyvitamin D and developed extensive calcification in soft tissues and vascular walls. Furthermore, compared with the Hyp mice, Hyp/klotho−/− mice were smaller in size, showed features of generalized tissue atrophy, and generally died by 15‐20 wk of age. Our in vivo studies provide genetic evidence for a pathological role of increased FGF23 activities in regulating abnormal phosphate homeostasis in Hyp mice. Moreover, these results suggest that even when serum levels of FGF23 are significantly high, in the absence of klotho, FGF23 is unable to regulate systemic phosphate homeostasis. Our in vivo observations have significant clinical implications in dis‐eases associated with increased FGF23 activity and suggest that the functions of FGF23 can be therapeu‐tically modulated by manipulating the effects of klotho.—Nakatani, Y., Ohnishi, M., Razzaque, M. S. Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model. FASEBJ. 23, 3702‐3711 (2009). www.fasebj.org

In vivo genetic evidence for suppressing vascular and soft tissue calcification through the reduction of serum phosphate levels, even in the presence of high serum calcium and 1,25-dihydroxyvitamin-D levels

Background— Klotho-knockout mice (klotho−/−) have increased renal expression of sodium/phosphate cotransporters (NaPi2a), associated with severe hyperphosphatemia. Such serum biochemical changes in klotho−/− mice lead to extensive soft-tissue anomalies and vascular calcification. To determine the significance of increased renal expression of the NaPi2a protein and concomitant hyperphosphatemia and vascular calcification in klotho−/− mice, we generated klotho and NaPi2a double-knockout (klotho−/−/NaPi2a−/−) mice. Methods and Results— Genetic inactivation of NaPi2a activity from klotho−/− mice reversed the severe hyperphosphatemia to mild hypophosphatemia or normophosphatemia. Importantly, despite significantly higher serum calcium and 1,25-dihydroxyvitamin D levels in klotho−/−/NaPi2a−/− mice, the vascular and soft-tissue calcifications were reduced. Extensive soft-tissue anomalies and cardiovascular calcification were consistently noted in klotho−/− mice by 6 weeks of age; however, these vascular and soft-tissue abnormalities were absent even in 12-week-old double-knockout mice. Klotho−/−/NaPi2a−/− mice also regained body weight and did not develop the generalized tissue atrophy often noted in klotho−/− single-knockout mice. Conclusion— Our in vivo genetic manipulation studies have provided compelling evidence for a pathological role of increased NaPi2a activities in regulating abnormal mineral ion metabolism and soft-tissue anomalies in klotho−/− mice. Notably, our results suggest that serum phosphate levels are the important in vivo determinant of calcification and that lowering serum phosphate levels can reduce or eliminate soft-tissue and vascular calcification, even in presence of extremely high serum calcium and 1,25-dihydroxyvitamin D levels. These in vivo observations have significant clinical importance and therapeutic implications for patients with chronic kidney disease with cardiovascular calcification.

CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci

The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct phenotype in colorectal cancer. However, the concept of CIMP-low with less extensive CpG island methylation is still evolving. Our aim is to examine whether density of methylation in individual CpG islands was different between CIMP-low and CIMP-high tumors. Utilizing MethyLight technology and 889 population-based colorectal cancers, we quantified DNA methylation (methylation index, percentage of methylated reference) at 14 CpG islands, including 8 CIMP-high-specific loci (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). Methylation positivity in each locus was defined as methylation index>4. Low-level methylation (methylation index>0, <20) in each CIMP-high-specific locus was significantly more common in 340 CIMP-low tumors (1/8–5/8 methylation-positive loci) than 133 CIMP-high tumors (≥6/8 methylation-positive loci) and 416 CIMP-0 tumors (0/8 methylation-positive loci) (P≤0.002). In the other six loci (CHFR, HIC1, IGFBP3, MGMT, MINT31 and WRN), which were not highly specific for CIMP-high, low-level methylation, was not persistently more prevalent in CIMP-low tumors. In conclusion, compared to CIMP-high and CIMP-0 tumors, CIMP-low colorectal cancers show not only few methylated CIMP-high-specific CpG islands, but also more frequent low-level methylation at individual loci. Our data may provide supporting evidence for a difference in pathogenesis of DNA methylation between CIMP-low and CIMP-high tumors.

Conversion of a Paracrine Fibroblast Growth Factor into an Endocrine Fibroblast Growth Factor

Background: The role of heparan sulfate (HS) in endocrine FGF signaling has not been defined. Results: Endocrine FGF mutants devoid of HS binding retain full metabolic activity. Conclusion: HS is dispensable for the metabolic activity of endocrine FGFs. Significance: The study provides new insights into the composition of the cell surface signaling complex of endocrine FGFs. FGFs 19, 21, and 23 are hormones that regulate in a Klotho co-receptor-dependent fashion major metabolic processes such as glucose and lipid metabolism (FGF21) and phosphate and vitamin D homeostasis (FGF23). The role of heparan sulfate glycosaminoglycan in the formation of the cell surface signaling complex of endocrine FGFs has remained unclear. Here we show that heparan sulfate is not a component of the signal transduction unit of FGF19 and FGF23. In support of our model, we convert a paracrine FGF into an endocrine ligand by diminishing heparan sulfate-binding affinity of the paracrine FGF and substituting its C-terminal tail for that of an endocrine FGF containing the Klotho co-receptor-binding site to home the ligand into the target tissue. In addition to serving as a proof of concept, the ligand conversion provides a novel strategy for engineering endocrine FGF-like molecules for the treatment of metabolic disorders, including global epidemics such as type 2 diabetes and obesity.

Klotho Coreceptors Inhibit Signaling by Paracrine Fibroblast Growth Factor 8 Subfamily Ligands

ABSTRACT It has been recently established that Klotho coreceptors associate with fibroblast growth factor (FGF) receptor tyrosine kinases (FGFRs) to enable signaling by endocrine-acting FGFs. However, the molecular interactions leading to FGF-FGFR-Klotho ternary complex formation remain incompletely understood. Here, we show that in contrast to αKlotho, βKlotho binds its cognate endocrine FGF ligand (FGF19 or FGF21) and FGFR independently through two distinct binding sites. FGF19 and FGF21 use their respective C-terminal tails to bind to a common binding site on βKlotho. Importantly, we also show that Klotho coreceptors engage a conserved hydrophobic groove in the immunoglobulin-like domain III (D3) of the “c” splice isoform of FGFR. Intriguingly, this hydrophobic groove is also used by ligands of the paracrine-acting FGF8 subfamily for receptor binding. Based on this binding site overlap, we conclude that while Klotho coreceptors enhance binding affinity of FGFR for endocrine FGFs, they actively suppress binding of FGF8 subfamily ligands to FGFR.

18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high

Background:The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.Methods:Utilizing MethyLight technology (real-time PCR), we quantified DNA methylation in 8 CIMP-specific promoters {CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1} in 758 non-MSI-high colorectal cancers obtained from two large prospective cohorts. Using four 18q microsatellite markers (D18S55, D18S56, D18S67 and D18S487) and stringent criteria for 18q LOH, we selected 374 tumors (236 LOH-positive tumors with ≥ 2 markers showing LOH; and 138 LOH-negative tumors with ≥ 3 informative markers and no LOH).Results:CIMP-0 (0/8 methylated promoters) was significantly more common in 18q LOH-positive tumors (59% = 139/236, p = 0.002) than 18q LOH-negative tumors (44% = 61/138), while CIMP-low/high (1/8–8/8 methylated promoters) was significantly more common (56%) in 18q LOH-negative tumors than 18q LOH-positive tumors (41%). These relations persisted after stratification by sex, location, or the status of MSI, p53 expression (by immunohistochemistry), or KRAS/BRAF mutation.Conclusion:18q LOH is correlated positively with CIMP-0 and inversely with CIMP-low and CIMP-high. Our findings provide supporting evidence for relationship between CIMP-0 and 18q LOH as well as a molecular difference between CIMP-0 and CIMP-low in colorectal cancer.