Chengliu Jin

FGFR4 Prevents Hyperlipidemia and Insulin Resistance but Underlies High Fat Diet-Induced Fatty Liver

OBJECTIVE—Fibroblast growth factor (FGF) family signaling largely controls cellular homeostasis through short-range intercell paracrine communication. Recently FGF15/19, 21, and 23 have been implicated in endocrine control of metabolic homeostasis. The identity and location of the FGF receptor isotypes that mediate these effects are unclear. The objective was to determine the role of FGFR4, an isotype that has been proposed to mediate an ileal FGF15/19 to hepatocyte FGFR4 axis in cholesterol homeostasis, in metabolic homeostasis in vivo. RESEARCH DESIGN AND METHODS—FGFR4−/− mice—mice overexpressing constitutively active hepatic FGFR4—and FGFR4−/− with constitutively active hepatic FGFR4 restored in the liver were subjected to a normal and a chronic high-fat diet sufficient to result in obesity. Systemic and liver-specific metabolic phenotypes were then characterized. RESULTS—FGFR4-deficient mice on a normal diet exhibited features of metabolic syndrome that include increased mass of white adipose tissue, hyperlipidemia, glucose intolerance, and insulin resistance, in addition to hypercholesterolemia. Surprisingly, the FGFR4 deficiency alleviated high-fat diet–induced fatty liver in obese mice, which is also a correlate of metabolic syndrome. Restoration of FGFR4, specifically in hepatocytes of FGFR4-deficient mice, decreased plasma lipid levels and restored the high-fat diet–induced fatty liver but failed to restore glucose tolerance and sensitivity to insulin. CONCLUSIONS—FGFR4 plays essential roles in systemic lipid and glucose homeostasis. FGFR4 activity in hepatocytes that normally serves to prevent systemic hyperlipidemia paradoxically underlies the fatty liver disease associated with chronic high-fat intake and obesity.

Differential Specificity of Endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in Complex with KLB

Background Recent studies suggest that betaKlotho (KLB) and endocrine FGF19 and FGF21 redirect FGFR signaling to regulation of metabolic homeostasis and suppression of obesity and diabetes. However, the identity of the predominant metabolic tissue in which a major FGFR-KLB resides that critically mediates the differential actions and metabolism effects of FGF19 and FGF21 remain unclear. Methodology/Principal Findings We determined the receptor and tissue specificity of FGF21 in comparison to FGF19 by using direct, sensitive and quantitative binding kinetics, and downstream signal transduction and expression of early response gene upon administration of FGF19 and FGF21 in mice. We found that FGF21 binds FGFR1 with much higher affinity than FGFR4 in presence of KLB; while FGF19 binds both FGFR1 and FGFR4 in presence of KLB with comparable affinity. The interaction of FGF21 with FGFR4-KLB is very weak even at high concentration and could be negligible at physiological concentration. Both FGF19 and FGF21 but not FGF1 exhibit binding affinity to KLB. The binding of FGF1 is dependent on where FGFRs are present. Both FGF19 and FGF21 are unable to displace the FGF1 binding, and conversely FGF1 cannot displace FGF19 and FGF21 binding. These results indicate that KLB is an indispensable mediator for the binding of FGF19 and FGF21 to FGFRs that is not required for FGF1. Although FGF19 can predominantly activate the responses of the liver and to a less extent the adipose tissue, FGF21 can do so significantly only in the adipose tissue and adipocytes. Among several metabolic and endocrine tissues, the response of adipose tissue to FGF21 is predominant, and can be blunted by the ablation of KLB or FGFR1. Conclusions Our results indicate that unlike FGF19, FGF21 is unable to bind FGFR4-KLB complex with affinity comparable to FGFR1-KLB, and therefore, at physiological concentration less likely to directly and significantly target the liver where FGFR4-KLB predominantly resides. However, both FGF21 and FGF19 have the potential to activate responses of primarily the adipose tissue where FGFR1-KLB resides.

Ectopic Activity of Fibroblast Growth Factor Receptor 1 in Hepatocytes Accelerates Hepatocarcinogenesis by Driving Proliferation and Vascular Endothelial Growth Factor–Induced Angiogenesis

Fibroblast growth factor (FGF) signaling mediates cell-to-cell communication in development and organ homeostasis in adults. Of the four FGF receptor (FGFR) tyrosine kinases, only FGFR4 is expressed in mature hepatocytes. Although FGFR1 is expressed by hepatic cell progenitors and adult nonparenchymal cells, ectopic expression is commonly observed in hepatoma cells. Here, we determined whether ectopic FGFR1 is a cause or consequence of hepatocellular carcinoma by targeting a constitutively active human FGFR1 to mouse hepatocytes. Livers of transgenic mice exhibited accelerated regeneration after partial hepatectomy but no signs of neoplastic or preneoplastic abnormalities for up to 18 months. However, in diethylnitrosamine-treated mice, the chronic FGFR1 activity promoted an incidence of 44% adenomas at 4 months and 38% hepatocellular carcinoma at 8 months. No adenoma or hepatocellular carcinoma was observed in diethylnitrosamine-treated wild-type (WT) livers at 4 or 8 months, respectively. At 10 and 12 months, tumor-bearing livers in transgenic mice were twice the size of those in WT animals. Isolated hepatoma cells from the transgenic tumors exhibited a growth advantage in culture. Advanced hepatocellular carcinoma in the transgenic livers exhibited a reduced rate of necrosis. This was accompanied by a mean microvessel density of 2.7 times that of WT tumors and a markedly higher level of vascular endothelial growth factor. In cooperation with an initiator, the persistent activity of ectopic FGFR1 in hepatocytes is a strong promoter of hepatocellular carcinoma by driving cell proliferation at early stages and promoting neoangiogenesis at late stages of progression.

Directionally specific paracrine communication mediated by epithelial FGF9 to stromal FGFR3 in two-compartment premalignant prostate tumors

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

Resident Hepatocyte Fibroblast Growth Factor Receptor 4 Limits Hepatocarcinogenesis

Fibroblast growth factor (FGF) family signaling mediates cell‐to‐cell communication in development and organ homeostasis in adults. Of the FGF receptor (FGFR) isotypes, FGFR4 is the sole resident isotype present in mature parenchymal hepatocytes. FGFR1 that is normally associated with activated nonparenchymal cells appears ectopically in hepatoma cells. Ectopic expression and chronic activity of FGFR1 in hepatocytes accelerates diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis by driving unrestrained cell proliferation and tumor angiogenesis. Hepatocyte FGFR4 mediates livers role in systemic cholesterol/bile acid and lipid metabolism and affects proper hepatolobular restoration after damage without effect on cell proliferation. Here we ask whether FGFR4 plays a role in progression of hepatocellular carcinoma (HCC). We report that although spontaneous HCC was not detected in livers of FGFR4‐deficient mice, the ablation of FGFR4 accelerated DEN‐induced hepatocarcinogenesis. In contrast to FGFR1 that induced a strong mitogenic response and depressed rate of cell death in hepatoma cells, FGFR4 failed to induce a mitogenic response and increased the rate of cell death. FGFR1 but not FGFR4 induced cyclin D1 and repressed p27 expression. Analysis of activation of Erk, JNK, and PI3K‐related AKT signaling pathways indicated that in contrast to FGFR1, FGFR4 failed to sustain Erk activation and did not activate AKT. These differences may underlie the opposing effects of FGFR1 and FGFR4. These results suggest that in contrast to ectopic FGFR1 that is a strong promoter of hepatoma, resident FGFR4 that mediates differentiated hepatocyte metabolic functions also serves to suppress hepatoma progression.

Metabolic Regulator βKlotho Interacts with Fibroblast Growth Factor Receptor 4 (FGFR4) to Induce Apoptosis and Inhibit Tumor Cell Proliferation

In organs involved in metabolic homeostasis, transmembrane α and βklothos direct FGFR signaling to control of metabolic pathways. Coordinate expression of βklotho and FGFR4 is a property of mature hepatocytes. Genetic deletion of FGFR4 or βklotho in mice disrupts hepatic cholesterol/bile acid and lipid metabolism. The deletion of FGFR4 has no effect on the proliferative response of hepatocytes after liver injury. However, its absence results in accelerated progression of dimethynitrosamine-initiated hepatocellular carcinomas, indicating that FGFR4 suppresses hepatoma proliferation. The mechanism underlying the FGFR4-mediated hepatoma suppression has not been addressed. Here we show that βklotho expression is more consistently down-regulated in human and mouse hepatomas than FGFR4. Co-expression and activation by either endocrine FGF19 or cellular FGF1 of the FGFR4 kinase in a complex with βklotho restricts cell population growth through induction of apoptotic cell death in both hepatic and nonhepatic cells. The βklotho-FGFR4 partnership caused a depression of activated AKT and mammalian target of rapamycin while activating ERK1/2 that may underlie the pro-apoptotic effect. Our results show that βklotho not only interacts with heparan sulfate-FGFR4 to form a complex with high affinity for endocrine FGF19 but also impacts the quality of downstream signaling and biological end points activated by either FGF19 or canonical FGF1. Thus the same βklotho-heparan sulfate-FGFR4 partnership that mediates endocrine control of hepatic metabolism plays a role in cellular homeostasis and hepatoma suppression through negative control of cell population growth mediated by pro-apoptotic signaling.

Self-renewal and multilineage differentiation of mouse dental epithelial stem cells

Understanding the cellular and molecular mechanisms underlying the self-renewal and differentiation of dental epithelial stem cells (DESCs) that support the unlimited growth potential of mouse incisors is critical for developing novel tooth regenerative therapies and unraveling the pathogenesis of odontogenic tumors. However, analysis of DESC properties and regulation has been limited by the lack of an in vitro assay system and well-documented DESC markers. Here, we describe an in vitro sphere culture system to isolate the DESCs from postnatal mouse incisor cervical loops (CLs) where the DESCs are thought to reside. The dissociated cells from CLs were able to expand and form spheres for multiple generations in the culture system. Lineage tracing indicated that DESC within the spheres were epithelial in origin as evident by lineage tracing. Upon stimulation, the sphere cells differentiated into cytokeratin 14- and amelogenin-expressing and mineral material-producing cells. Compared to the CL tissue, sphere cells expressed high levels of expression of Sca-1, CD49f (also designated as integrin α6), and CD44. Fluorescence-activated cell sorting (FACS) analyses of mouse incisor CL cells further showed that the CD49f(Bright) population was enriched in sphere-forming cells. In addition, the CD49f(Bright) population includes both slow-cycling and Lgr5(+) DESCs. The in vitro sphere culture system and identification of CD49f(Bright) as a DESC marker provide a novel platform for enriching DESCs, interrogating how maintenance, cell fate determination, and differentiation of DESCs are regulated, and developing tooth regenerative therapies.

Novel phosphotyrosine targets of FGFR2IIIb signaling.

In partnership exclusively with the epithelial FGFR2IIIb isotype and a structurally-specific heparan sulfate motif, stromal-derived FGF7 delivers both growth-promoting and growth-limiting differentiation signals to epithelial cells that promote cellular homeostasis between stromal and epithelial compartments. Intercompartmental homeostasis supported by FGF7/FGFR2IIIb is subverted in many solid epithelial tumors. The normally mesenchymal-derived homologue FGFR1 drives proliferation and a progressive tumor-associated phenotype when it appears ectopically in epithelial cells. In order to understand the mechanism underlying the unique biological effects of FGFR2IIIb, we developed an inducible FGFR2IIIb expression system that is specifically dependent on FGF7 for activation in an initially unresponsive cell line to avoid selection for only the growth-promoting aspects of FGFR2IIIb signaling. We then determined FGF7/FGFR2IIIb signaling-specific tyrosine phosphorylated proteins within 5 min after FGF7 stimulation by phosphopeptide immunoaffinity purification and nano-LC-MS/MS. The FGF7/FGFR2 pair caused tyrosine phosphorylation of multiple proteins that have been implicated in the growth stimulating activities of FGFR1 that included multi-substrate organizers FRS2alpha and IRS4, ERK2 and phosphatases SHP2 and SHIP2. It uniquely phosphorylated CDK2 and phosphatase PTPN18 on sites involved in the attenuation of cell proliferation, and several factors that maintain nuclear-cytosolic relationships (emerin and LAP2), protein structure and other cellular fine structures as well as some proteins of unknown functions. Several of the FGF7/FGFR2IIIb-specific targets have been associated with maintenance of function and tumor suppression and disruption in tumors. In contrast, a number of pTyr substrates associated with FGF2/FGFR1 that are generally associated with intracellular Ca(2+)-phospholipid signaling, membrane and cytoskeletal plasticity, cell adhesion, migration and the tumorigenic phenotype were not observed with FGF7/FGFR2IIIb. Our findings provide specific downstream targets for dissection of causal relationships underlying the distinct role of FGF7/FGFR2IIIb signaling in epithelial cell homeostasis.

Control of lipid metabolism by adipocyte FGFR1-mediated adipohepatic communication during hepatic stress

BackgroundEndocrine FGF19 and FGF21 exert their effects on metabolic homeostasis through fibroblast growth factor receptor (FGFR) and co-factor betaKlotho (KLB). Ileal FGF19 regulates bile acid metabolism through specifically FGFR4-KLB in hepatocytes where FGFR1 is not significant. Both FGF19 and FGF21 activate FGFR1-KLB whose function predominates in adipocytes. Recent studies using administration of FGF19 and FGF21 and genetic ablation of KLB or adipocyte FGFR1 indicate that FGFR1-KLB mediates the response of adipocytes to both FGF21 and FGF19. Here we show that adipose FGFR1 regulates lipid metabolism through direct effect on adipose tissue and indirect effects on liver under starvation conditions that cause hepatic stress.MethodsWe employed adipocyte-specific ablations of FGFR1 and FGFR2 genes in mice, and analyzed metabolic consequences in adipose tissue, liver and systemic parameters under normal, fasting and starvation conditions.ResultsUnder normal conditions, the ablation of adipose FGFR1 had little effect on adipocytes, but caused shifts in expression of hepatic genes involved in lipid metabolism. Starvation conditions precipitated a concurrent elevation of serum triglycerides and non-esterified fatty acids, and increased hepatic steatosis and adipose lipolysis in the FGFR1-deficient mice. Little effect on glucose or ketone bodies due to the FGFR1 deficiency was observed.ConclusionsOur results suggest an adipocyte-hepatocyte communication network mediated by adipocyte FGFR1 that concurrently dampens hepatic lipogenesis and adipocyte lipolysis. We propose that this serves overall to mete out and extend lipid reserves for neural fuels (glucose and ketone bodies), while at the same time governing extent of hepatosteatosis during metabolic extremes and other conditions causing hepatic stress.

Fibroblast growth factor signaling is essential for self-renewal of dental epithelial stem cells

Background: Understanding of the self-renewal and differentiation of dental epithelial stem cells (DESCs) is important for tooth regeneration therapies. Results: Depletion of FGF signaling suppressed self-renewal and led to differentiation of DESCs. Conclusion: FGF signaling is essential for maintenance of DESCs. Significance: The finding sheds new light on the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated. A constant supply of epithelial cells from dental epithelial stem cell (DESC) niches in the cervical loop (CL) enables mouse incisors to grow continuously throughout life. Elucidation of the cellular and molecular mechanisms underlying this unlimited growth potential is of broad interest for tooth regenerative therapies. Fibroblast growth factor (FGF) signaling is essential for the development of mouse incisors and for maintenance of the CL during prenatal development. However, how FGF signaling in DESCs controls the self-renewal and differentiation of the cells is not well understood. Herein, we report that FGF signaling is essential for self-renewal and the prevention of cell differentiation of DESCs in the CL as well as in DESC spheres. Inhibiting the FGF signaling pathway decreased proliferation and increased apoptosis of the cells in DESC spheres. Suppressing FGFR or its downstream signal transduction pathways diminished Lgr5-expressing cells in the CL and promoted cell differentiation both in DESC spheres and the CL. Furthermore, disruption of the FGF pathway abrogated Wnt signaling to promote Lgr5 expression in DESCs both in vitro and in vivo. This study sheds new light on understanding the mechanism by which the homeostasis, expansion, and differentiation of DESCs are regulated.