George B. John

Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse

Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals.

Klotho Inhibits Transforming Growth Factor-β1 (TGF-β1) Signaling and Suppresses Renal Fibrosis and Cancer Metastasis in Mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.

Foxo3 is a PI3K-dependent molecular switch controlling the initiation of oocyte growth

In mammals, oocytes are packaged into compact structures-primordial follicles-which remain inert for prolonged intervals until individual follicles resume growth via a process known as primordial follicle activation. Here we show that the phosphoinositide 3-kinase (PI3K) signalling pathway controls primordial follicle activation through the forkhead transcription factor Foxo3. Within oocytes, Foxo3 is regulated by nucleocytoplasmic shuttling. Foxo3 is imported into the nucleus during primordial follicle assembly, and is exported upon activation. Oocyte-specific ablation of Pten resulted in PI3K-induced Akt activation, Foxo3 hyperphosphorylation, and Foxo3 nuclear export, thereby triggering primordial follicle activation, defining the steps by which the PI3K pathway and Foxo3 control this process. Inducible ablation of Pten and Foxo3 in adult oocytes using a new tool for genetic analysis of the germline, Vasa-Cre(ERT2), showed that this pathway functions throughout life. Thus, a principal physiologic role of the PI3K pathway is to control primordial follicle activation via Foxo3.

Role of Klotho in Aging, Phosphate Metabolism, and CKD

The klotho gene (KL) was identified first as a putative aging-suppressor gene that extended life span when overexpressed and accelerated aging-like phenotypes when disrupted in mice. It encodes a single-pass transmembrane protein and is expressed predominantly in kidney, where it functions as an obligate coreceptor for fibroblast growth factor 23 (FGF-23). FGF-23 is a bone-derived hormone that suppresses phosphate reabsorption and 1,25 dihydroxyvitamin D(3) (vitamin D) synthesis in the kidney. Klotho also is expressed in the parathyroid gland, where FGF-23 decreases parathyroid hormone expression and secretion, further suppressing vitamin D synthesis in kidney. Thus, FGF-23 functions as a phosphaturic hormone and a counter-regulatory hormone for vitamin D, thereby inducing negative phosphate balance. Mice lacking either FGF-23 or Klotho show hyperphosphatemia in addition to developing multiple aging-like phenotypes, which can be rescued by resolving phosphate retention. These findings have unveiled an unexpected link between aging and phosphate. In patients with chronic kidney disease (CKD), phosphate retention is seen universally and has been associated with increased mortality risk. Patients with CKD have high serum FGF-23 levels with decreased klotho expression in the kidney and parathyroid, rendering FGF-23 and Klotho as potential biomarkers and therapeutic targets for CKD. The Klotho protein not only serves as a coreceptor for FGF-23, but also functions as a humoral factor. Klothos extracellular domain is released into blood and urine by ectodomain shedding and exerts various functions independently of FGF-23, including regulation of multiple ion channels and transporters. Decreased urinary Klotho protein level has been identified as one of the earliest biomarkers of CKD progression. This review focuses on the current understanding of Klotho protein function, with emphasis on its potential involvement in the pathophysiologic process of CKD.

Specificity of the Requirement for Foxo3 in Primordial Follicle Activation

Primordial follicles are long-lived structures assembled early in life. The mechanisms that control the balance between the conservation and the activation of primordial follicles are critically important for fertility and dictate the onset of menopause. The forkhead transcription factor Foxo3 serves an essential role in these processes by suppressing the growth of primordial follicles, thereby preserving them until later in life. While other factors regulating primordial follicle growth have been described, most serve multiple functions at several stages of female germ cell or follicle development, and corresponding mouse mutants exhibit pleiotropic phenotypes with disruption of multiple stages of follicle assembly, development, or survival. To investigate the possibility that Foxo3 also functions in other aspects of ovarian development beyond its known role in primordial follicle activation (PFA), we performed detailed analyses of mouse ovaries including electron microscopy to study primordial follicle structure, assembly, and early growth. These analyses revealed that the timing of primordial follicle assembly, early oocyte survival, and the expression of early germ line markers were unaffected in early Foxo3 ovaries. Taken together, these studies demonstrate that the phenotype associated with Foxo3 deficiency is remarkably specific for PFA and further support the placement of Foxo3 in a unique phenotypic class among mammalian female sterile mutants. Lastly, we discuss the implications of the specificity of this mutant phenotype with regard to the hypothesis that oocyte regeneration may occur in adults and serves as a means to replenish oocytes lost via natural physiological processes.

Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse

Female infertility syndromes are among the most prevalent chronic health disorders in women, but their genetic basis remains unknown because of uncertainty regarding the number and identity of ovarian factors controlling the assembly, preservation, and maturation of ovarian follicles. To systematically discover ovarian fertility genes en masse, we employed a mouse model (Foxo3) in which follicles are assembled normally but then undergo synchronous activation. We developed a microarray-based approach for the systematic discovery of tissue-specific genes and, by applying it to Foxo3 ovaries and other samples, defined a surprisingly large set of ovarian factors (n = 348, ∼1% of the mouse genome). This set included the vast majority of known ovarian factors, 44% of which when mutated produce female sterility phenotypes, but most were novel. Comparative profiling of other tissues, including microdissected oocytes and somatic cells, revealed distinct gene classes and provided new insights into oogenesis and ovarian function, demonstrating the utility of our approach for tissue-specific gene discovery. This study will thus facilitate comprehensive analyses of follicle development, ovarian function, and female infertility.

Kit Signaling via PI3K promotes ovarian follicle maturation but is dispensable for primordial follicle activation

In mammals, primordial follicles are generated early in life and remain dormant for prolonged intervals. Their growth resumes via a process known as primordial follicle activation. Recent genetic studies have demonstrated that phosphoinositide 3-kinase (PI3K) is the essential signaling pathway controlling this process throughout life, acting via Akt to regulate nucleocytoplasmic shuttling of Foxo3, which functions as a downstream molecular switch. The receptor tyrosine kinase Kit has been implicated by numerous studies as the critical upstream regulator of primordial follicle activation via PI3K/Akt. Here we present a genetic analysis of the contribution of Kit in regulating primordial follicle activation and early follicle growth, employing a knock-in mutation (Kit(Y719F)) that completely abrogates signaling via PI3K. Surprisingly, homozygous Kit(Y719F) female mice undergo primordial follicle activation and are fertile, demonstrating that Kit signaling via PI3K is dispensable for this process. However, other abnormalities were identified in Kit(Y719F) ovaries, including accelerated primordial follicle depletion and accumulation of morphologically abnormal primary/secondary follicles with persistent nuclear Foxo3 localization. These findings reveal specific roles of Kit in the maintenance of the primordial follicle reserve and in the primary to secondary follicle transition, but argue that Kit is dispensable in primordial follicle activation.

Prospective Analysis of Ki-67 as an Independent Predictor of Oncologic Outcomes in Patients with High Grade Upper Tract Urothelial Carcinoma

PURPOSE We determined the association of the proliferation marker Ki-67 with pathological parameters and oncologic outcomes in patients with high grade upper tract urothelial carcinoma. MATERIALS AND METHODS Immunohistochemical staining for Ki-67 was done prospectively in 101 consecutive patients undergoing radical nephroureterectomy/ureterectomy for high grade upper tract urothelial carcinoma. Data were compared based on Ki-67 status (normal vs over expressed). Survival was assessed by the Kaplan-Meier method. Cox regression analysis was done to identify independent predictors of time dependent outcomes. RESULTS Median patient age was 70.0 years and median followup was 22.0 months (range 1 to 77). Overall, 30.2% of the population experienced recurrence and 24.8% died of upper tract urothelial carcinoma. Organ confined disease (T2 or less and lymph node negative), lymphovascular invasion and sessile architecture were present in 56.3%, 33.3% and 20.8% of patients, respectively. Ki-67 was over expressed in 73.3% of patients and associated with adverse pathological features. Patients with over expressed Ki-67 had significantly worse recurrence-free survival (43.2 vs 69.0 months, p = 0.006) and cancer specific survival (48.9 vs 68.9 months, p = 0.031) than patients with normal Ki-67. Patients with nonmetastatic disease similarly had worse recurrence-free survival (40.7 vs 71.8 months, p = 0.003) and cancer specific survival (41 months vs not attained, p = 0.008) for over expressed vs normal Ki-67. After adjusting for the effects of organ vs nonorgan confined disease Ki-67 over expression was an independent predictor of recurrence-free survival in the total cohort (HR 4.3, p = 0.05) and in patients with nonmetastatic disease (HR 8.5, p = 0.038). CONCLUSIONS Ki-67 over expression was associated with adverse pathological features in cases of upper tract urothelial carcinoma. It was also an independent predictor of recurrence-free survival in patients with high grade upper tract urothelial carcinoma.

The anatomical basis for wrinkles

BACKGROUND Light and electron microscopy have not identified a distinct anatomical structure associated with either skin wrinkles or creases, and a histological difference between wrinkled and adjacent skin has not been identified. OBJECTIVES The authors investigate whether facial wrinkles are related to underlying lymphatic vessels and perilymphatic fat. METHODS Lymphatic vessels with a specialized tube of perilymphatic fat were identified beneath palmar creases. Sections of skin, adipose tissue, and muscle were harvested from each of 13 cadavers. Three sites were investigated: the transverse forehead crease, lateral orbicularis oculi wrinkle (crows feet), and the nasojugal crease. The tissue was paraffin embedded and processed. Two-step indirect immunohistochemistry was performed, and images were examined using laser confocal microscopy. Measurements were taken with software. RESULTS Every wrinkle examined was found above and within ±1 mm of a major lymphatic vessel and its surrounding tube of adipose tissue. The results satisfied our null hypothesis and were statistically significant. Lymphatic vessels were identified by positive immunofluorescence as well as histological criteria. These findings have been further validated by fluorochrome tracer studies. CONCLUSIONS An anatomical basis for wrinkles was identified among the specimens studied. Lymphatic vessels, along with the surrounding distinct perilymphatic fat, traveled directly beneath wrinkles and creases. Lymphatic dysregulation leads to inflammation, scarring, and fibrosis, but inadvertent injection of these vessels can be avoided with anatomical knowledge.