The discovery of alpha-Klotho and FGF23 unveiled new insight into calcium and phosphate homeostasis.

1The traditional view of calcium homeostasis is that it is maintained by two essential reactions. First, changes in extracellular Ca2+ are sensed in several distinct cell types, stimulating the secretion of parathyroid hormone (PTH), 1,25(OH)(2) D and calcitonin in response to the body's requirement. Second, these calcitropic hormones then act on the calcium-translocating cells of the kidney, bone, and intestine to restore calcium balance. Recent progress indicates that alpha-Klotho and fibroblast growth factor (FGF) 23 are key players that integrate the multi-step regulatory system of calcium homeostasis that rapidly adjusts the extracellular calcium concentration and continuously maintains its concentration within a narrow physiological range. alpha-Klotho and FGF23 are also found to be major players in the regulatory system of phosphate homeostasis. Here, the demonstration of the molecular functions of alpha-Klotho and FGF23 has recently given new insight into the field of calcium and phosphate homeostasis.

Defects in growth and bone metabolism in klotho mutant mice are resistant to GH treatment.

Klotho mutant (kl/kl) mice exhibit growth retardation after weaning, and previous electron microscopic examination of GH-producing cells in pituitary glands revealed a reduction in GH granules. However, it has not been known whether growth retardation in klotho mutant mice is related to the loss of GH function. We therefore examined whether treatment with GH could rescue the retardation of growth. At the end of 3 weeks of treatment with human GH, the body weight of wild-type (WT) mice was increased. In contrast, body weight was not increased in klotho mutant mice even after the treatment with human GH. Another feature of klotho mutant mice is the presence of osteopetrosis in the epiphyses of long bones and vertebrae. Treatment with human GH increased trabecular bone volume in the epiphyseal region of WT tibiae. Interestingly, increase in trabecular bone volume by GH treatment was also observed in klotho mutant mice and, therefore, the phenotype of high bone volume in the klotho mice was further enhanced. These findings indicate that a GH receptor system in cancellous bones could operate in mutant mice. Thus, growth retardation in the klotho mutant mice is resistant against GH treatment even when these mice respond to GH treatment in terms of cancellous bone volume.

Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord.

Recent studies have demonstrated that neural stem cells and other progenitors are present in the adult CNS. Details of their properties, however, remain poorly understood. Here we examined the properties and control mechanisms of neural progenitors in the adult rat spinal cord at the molecular level. Adult and embryonic progenitors commonly expressed various homeodomain-type (Pax6, Pax7, Nkx2.2, and Prox1) and basic helix-loop-helix (bHLH)-type (Ngn2, Mash1, NeuroD1, and Olig2) transcriptional regulatory factors in vitro. Unlike their embryonic counterparts, however, adult progenitors could not generate specific neurons that expressed markers appropriate for spinal motoneurons or interneurons, including Islet1, Lim1, Lim3, and HB9. Cells expressing the homeodomain factors Pax6, Pax7, and Nkx2.2 also emerged in vivo in response to injury and were distributed in unique patterns in the lesioned spinal cord. However, neither the expression of the neurogenic bHLH factors including Ngn2, Mash1, and NeuroD1 nor subsequent generation of new neurons could be detected in injured tissue. Our results suggest that signaling through the cell-surface receptor Notch is involved in this restriction. The expression of Notch1 in vivo was enhanced in response to injury. Furthermore, activation of Notch signaling in vitro inhibited differentiation of adult progenitors, whereas attenuation of Notch signals and forced expression of Ngn2 significantly enhanced neurogenesis. These results suggest that both the intrinsic properties of adult progenitors and local environmental signals, including Notch signaling, account for the limited regenerative potential of the adult spinal cord.

The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc.

Reduction in klotho gene expression causes accelerated senescence in klotho mutant mice. We have now found two key substances, phosphorus and zinc, which affect the appearance of klotho phenotypes. Klotho mutant homozygotes fed nonpurified diet with a phosphorus concentration of 1.03 g/100 g showed typical klotho phenotypes. However, most of the klotho phenotypes no longer appeared in male homozygotes fed a 0.4 g/100 g phosphorus diet. These homozygotes were capable of spermatogenesis. In the kidneys of the rescued male homozygotes, klotho protein expression was clearly detected. On the other hand, female klotho mice required supplementation of 0.25 g/100 g zinc orotate to the 0.4 g/100 g phosphorus diet to be rescued. Unlike in the rescued male mice, klotho protein levels in the kidneys of the rescued females were quite low. Wild-type (C3H/He) mice fed 1.5 or 1.0 g/100 g phosphorus diets had lower klotho protein expression in the kidneys than those fed a 0.4 g/100 g phosphorus diet (Kruskal-Wallis test, P < 0.05). These results indicate that dietary phosphorus and zinc modulate the phenotypes of klotho mice, and that klotho expression in the kidneys is regulated not only in klotho mutant mice, but also in wild-type mice.

Expression of Na+/Ca(2+) exchanger (NCX1) gene in the developmental mouse embryo and adult mouse brain.

The Na(+)/Ca(2+) exchanger gene, NCX1, is widely expressed in many tissues, encoding several isoforms through alternative RNA splicing. NCX1 deficient mice are known to be lethal at embryonic day 9-10 (E9-10). However, its expression pattern during embryogenesis is largely unknown. Therefore, to identify and compare the localization and alternatively spliced isoforms of NCX1 mRNA expressed in the developmental stages, we analyzed the mouse embryo. Northern blot analysis demonstrated that NCX1 mRNA was expressed from the earliest stage examined, E7. In situ hybridization analysis revealed that NCX1 mRNA was expressed in the heart alone until E10.5. However, at E14.5 and 16.5, NCX1 mRNA was expressed not only in the heart, but also in neuronal cells. In addition, the expression of NCX1 mRNA in the adult brain was most abundant in the hippocampus. Using reverse transcription-polymerase chain reaction (RT-PCR), we also identified the alternatively spliced isoforms expressed during each developmental stage. The restricted expression of the NCX1 gene suggested that NCX1 may play an important role in the developing mouse embryo.

Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons.

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. Our results further revealed crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. We suggest that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube.

Neurofilaments of Klotho, the mutant mouse prematurely displaying symptoms resembling human aging.

We reported previously that neurofilaments (NFs) of aged rats were highly packed in the axon and contained a smaller amount of NF-M as compared with those of young rats (Uchida et al. [1999] J. Neurosci. Res. 58:337-348). We studied NFs of the mutant mouse, named Klotho, which displays prematurely symptoms resembling human aging. The transport of axonal cytoskeletal proteins, including NFs, tubulin and actin, was decreased at the leading portion of the peak of transported proteins in Klotho during the process of premature aging. The nearest neighbor inter-NF distance in Klotho axons (35-39 nm) was shorter than that of the wild-type mouse (48-49 nm), indicating the packing of NFs in Klotho. The ratio of NF-M to NF-L was slightly decreased in cytoskeletons from the spinal cords of Klotho. These changes are similar, though not identical, to those observed in aged rats, and are the first evidence of age-related changes in the neurons of Klotho.

Klotho-deficient mice are resistant to bone loss induced by unloading due to sciatic neurectomy.

Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.

Sgn1, a basic helix-loop-helix transcription factor delineates the salivary gland duct cell lineage in mice.

The salivary system in mammals is comprised of three independently developed pairs of organs, the parotid, submaxillar, and sublingual glands. Each gland is composed of various ductal and acinar cell types that fulfill multiple roles. However, the molecular mechanisms regulating their biogenesis and functions are still largely unknown. In this paper, we report that two class B basic helix-loop-helix (bHLH) transcriptional regulators delineate the ductal and the acinar cells in salivary glands. Sgn1, a novel class B bHLH factor, is specifically expressed in the salivary duct cells, while the acinar cells are characterized by the expression of another class B bHLH factor, Mist1. The molecular nature of Sgn1 was also investigated: it binds to specific sequences of DNA as a dimer with a class A bHLH factor and acts as a negative transcriptional regulator against other bHLH factors. This study provides an important cue towards better understanding of the generation and function of multiple cell types in salivary glands. In addition, Sgn1 expression exhibits a reverse relationship with the development of male phenotypes, suggesting its role in gender dimorphism in the salivary glands.

Troglitazone improves endothelial function and augments renal klotho mRNA expression in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with multiple atherogenic risk factors.

Targeted disruption of the klotho gene induces multiple phenotypes characteristic of human aging, including arteriosclerosis, pulmonary emphysema and osteoporosis. Moreover, we previously observed that insufficient klotho expression in mice leads to endothelial dysfunction. In the present study, we used Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which exhibit hypertension, obesity, severe hyperglycemia and hypertriglyceridemia, and are thus considered an animal model of atherogenic disease, to test the effects of oral administration of troglitazone (200 mg/kg) on renal klotho mRNA expression and endothelial function. Systolic blood pressure, body weight, plasma glucose and triglyceride levels were all significantly higher in 30-week-old OLETF rats than in controls (LETO; Long-Evans Tokushima Otsuka) (p<0.05, n=7). In addition, endothelium-dependent relaxation of the aorta in response to 10(-5) M acetylcholine was significantly attenuated in OLETF rats (p<0.05, n=7), as was renal expression of klotho mRNA. Administration of troglitazone for 10 weeks significantly reduced systolic blood pressure, plasma glucose and triglyceride levels in OLETF rats, while augmenting endothelium-dependent aortic relaxation and renal klotho mRNA expression. These findings suggest that troglitazone protects the vascular endothelium against damage caused by the presence of multiple atherogenic factors.

Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3.

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.

Disruption of klotho gene causes an abnormal energy homeostasis in mice.

klotho mice, which genetically lack klotho gene expression, are characterized with various systemic phenotypes resembling human aging, and also with growth retardation. Here we show that klotho mice have a barely detectable amount of the white adipose tissue but their brown adipose tissue (BAT) is comparably preserved. Glucose tolerance and insulin sensitivity in klotho mice are increased compared to those in wild-type mice as revealed by intraperitoneal glucose and insulin tolerance tests. Uncoupling protein-1 gene expression of BAT and body temperature in klotho mice are lower than those in wild-type mice, suggesting that klotho mice have less energy expenditure than wild-type mice. Histological examination suggests that klotho mice possess less energy storage than wild-type mice with respect to glycogen in the liver and lipid in BAT. All these changes of parameters for energy homeostasis in klotho mice are very similar to those reported under food-restricted conditions. However, the amount of food intake is not different between klotho and wild-type mice when normalized for body weight. The present study elucidates the importance of klotho gene expression for the maintenance of normal energy homeostasis.

Decreased insulin production and increased insulin sensitivity in the klotho mutant mouse, a novel animal model for human aging.

We have recently identified a novel gene, klotho (kl), which may suppress several aging phenotypes. A defect of kl gene expression in the mouse results in a syndrome resembling human aging, such as arteriosclerosis, skin atrophy, osteoporosis, and pulmonary emphysema. To determine whether mouse homozygotes for the kl mutation (kl/kl) show abnormal glucose metabolism, an oral glucose tolerance test (OGTT) was performed at 6 to 8 weeks of age. Blood glucose levels during the OGTT were significantly lower in kl/kl mice versus wild-type mice. The insulin content of the pancreas was significantly lower in kl/kl mice compared with wild-type mice. Decreased insulin production was also supported by Northern blot analysis showing lower levels of insulin mRNA in kl/kl mice. To examine how lower blood glucose levels may exist in kl/kl mice despite decreased insulin production, insulin tolerance tests (ITTs) were performed. The glucose decline following insulin injection was more severe in kl/kl mice versus wild-type mice, suggesting that insulin sensitivity was higher in kl/kl mice versus wild-type mice. In kl/kl mice, an augmented expression of GLUT4 in skeletal muscle was demonstrated by both Northern blot analysis and Western blot analysis. Thus, we conclude that insulin production is decreased and insulin sensitivity is increased in the klotho mouse, a novel animal model for human aging.

In vivo klotho gene delivery protects against endothelial dysfunction in multiple risk factor syndrome.

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes (e.g., arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, and short life span). We have previously shown that mice heterozygous for a defect in the klotho gene upon parabiosis with wild-type mice show improved endothelial function, suggesting that the klotho gene product protects against endothelial dysfunction. In the present study, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat which demonstrates multiple atherogenic risk factors (e.g., hypertension, obesity, severe hyperglycemia, and hypertriglyceridemia) and is thus considered an experimental animal model of atherosclerotic disease, we show that adenovirus-mediated klotho gene delivery can (1) ameliorate vascular endothelial dysfunction, (2) increase nitric oxide production, (3) reduce elevated blood pressure, and (4) prevent medial hypertrophy and perivascular fibrosis. Based on these findings, klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function (e.g., hypertension and vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.

Ets family transcription factor ESE-1 is expressed in corneal epithelial cells and is involved in their differentiation.

Involvement of an epithelium-specific transcription factor ESE-1/ESX/ELF3/jen (ESE-1) in corneal epithelial cell differentiation was investigated. ESE-1 was reported to be induced during terminal differentiation of the epidermis and primary keratinocytes and to transactivate target genes through ets binding sites. However, its expression and function in corneal epithelium have not been examined. We report here that ESE-1 is upregulated upon differentiation in mouse corneal epithelium and in immortalized human corneal epithelial cells (HCE). ESE-1 transactivates through the regulatory element of cornea-specific K12 keratin. Moreover, introduction of ESE-1 antisense RNA in HCE cells affect their differentiation. These data suggest the involvement of ESE-1 in differentiation of corneal epithelial cells.

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Molecular cloning and expression of a novel klotho-related protein.

Klotho protein is a novel beta-glucosidase-like protein produced predominantly in the kidney. The klotho mouse, which genetically lacks klotho gene expression, manifests various systemic phenotypes resembling aging. In the present study we succeeded in isolating a novel human protein structurally related to klotho protein. The protein possesses one beta-glucosidase-like domain and is 42% identical with klotho protein at the amino acid level. Unlike klotho protein, it possesses neither a signal sequence nor a transmembrane domain, suggesting that it is a cytosolic protein, and thus was termed cytosolic beta-glucosidase-like protein-1 (cBGL1). By Northern blot analysis cBGL1 mRNA was expressed most abundantly in the liver, followed by the small intestine, colon, spleen, and kidney. When klotho and cBGL1 gene expression was examined in renal cell carcinoma tissues, both klotho and cBGL1 mRNA levels in tumors were lower than those in nontumor regions, suggesting that renal epithelial cells may lose klotho and cBGL1 gene expression during the course of malignant transformation. In conclusion, we describe the primary structure and gene expression of a novel protein related to klotho protein.