MicroRNA-<em>21</em> (miR-<em>21</em>) was reported to be overexpressed and contributes to invasion and gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to evaluate whether miR-<em>21</em> expression was associated with the overall survival (OS) of PDAC patients treated with gemcitabine and to provide mechanistic insights for new therapeutic targets. miR-<em>21</em> expression was evaluated in cells (including 7 PDAC cell lines, 7 primary cultures, <em>fibroblasts</em>, and a normal pancreatic ductal cell line) and tissues (neoplastic specimens from 81 PDAC patients and normal ductal samples) isolated by laser microdissection. The role of miR-<em>21</em> on the pharmacologic effects of gemcitabine was studied with a specific miR-<em>21</em> precursor (pre-miR-<em>21</em>). Patients with high miR-<em>21</em> expression had a significantly shorter OS both in the metastatic and in the adjuvant setting. Multivariate analysis confirmed the prognostic significance of miR-<em>21</em>. miR-<em>21</em> expression in primary cultures correlated with expression in their respective tissues and with gemcitabine resistance. Pre-miR-<em>21</em> transfection significantly decreased antiproliferative effects and apoptosis induction by gemcitabine, whereas matrix metalloproteinase (MMP)-2/MMP-9 and vascular endothelial <em>growth</em> <em>factor</em> expression were upregulated. Addition of inhibitors of phosphoinositide 3-kinase and mammalian target of rapamycin resulted in decrease of phospho-Akt and prevented pre-miR-<em>21</em>-induced resistance to the proapoptotic effects of gemcitabine. miR-<em>21</em> expression correlated with outcome in PDAC patients treated with gemcitabine. Modulation of apoptosis, Akt phosphorylation, and expression of genes involved in invasive behavior may contribute to the role of miR-<em>21</em> in gemcitabine chemoresistance and to the rational development of new targeted combinations.

The ability of heparin or that of hexuronyl hexosaminoglycan sulfate (HHS-4) to protect basic or acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the <em>growth</em> of baby hamster kidney (BHK-<em>21</em>) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at -80 degrees C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65 degrees C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS-4, basic and acidic FGF retained their potency. The effect of heparin and HHS-4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS-4 concentrations. This indicates that heparin and HHS-4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.

Bone marrow-Derived cells have been proposed to form new vessels or at least incorporate into <em>growing</em> vessels in adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow-Derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) and tumor <em>growth</em>. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells from littermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation, the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelial <em>growth</em> <em>factor</em> or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Seven and <em>21</em> days after surgery, proximal hindlimb muscles with <em>growing</em> collateral arteries and ischemic gastrocnemius muscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFP signals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocal microscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells. Nevertheless, we observed accumulations of GFP-positive cells around <em>growing</em> collateral arteries (3-fold increase versus nonoccluded side, P<0.001) and in ischemic distal hindlimbs. These cells were identified as <em>fibroblasts</em>, pericytes, and primarily leukocytes that stained positive for several <em>growth</em> <em>factors</em> and chemokines. Our findings suggest that in the adult organism, bone marrow-Derived cells do not promote vascular <em>growth</em> by incorporating into vessel walls but may function as supporting cells.

We review the physiology and pharmacology of two atypical fibroblast growth factors (FGFs)-FGF15/19 and FGF21-that can function as hormones. Both FGF15/19 and FGF21 act on multiple tissues to coordinate carbohydrate and lipid metabolism in response to nutritional status. Whereas FGF15/19 is secreted from the small intestine in response to feeding and has insulin-like actions, FGF21 is secreted from the liver in response to extended fasting and has glucagon-like effects. FGF21 also acts in an autocrine fashion in several tissues, including adipose. The pharmacological actions of FGF15/19 and FGF21 make them attractive drug candidates for treating metabolic disease.

There appear to be 2 pathways involved in the early pathogenesis of premalignant monoclonal gammopathy of undetermined significance (MGUS) and malignant multiple myeloma (MM) tumors. Nearly half of these tumors are nonhyperdiploid and mostly have immunoglobulin H (IgH) translocations that involve 5 recurrent chromosomal loci, including 11q13 (cyclin D1), 6p<em>21</em> (cyclin D3), 4p16 (<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 [FGFR3] and multiple myeloma SET domain [MMSET]), 16q23 (c-maf), and 20q11 (mafB). The remaining tumors are hyperdiploid and contain multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and <em>21</em>, but infrequently have IgH translocations involving the 5 recurrent loci. Dysregulated expression of cyclin D1, D2, or D3 appears to occur as an early event in virtually all of these tumors. This may render the cells more susceptible to proliferative stimuli, resulting in selective expansion as a result of interaction with bone marrow stromal cells that produce interleukin-6 (IL-6) and other cytokines. There are 5 proposed tumor groups, defined by IgH translocations and/or cyclin D expression, that appear to have differences in biologic properties, including interaction with stromal cells, prognosis, and response to specific therapies. Delineation of the mechanisms mediating MM cell proliferation, survival, and migration in the bone marrow (BM) microenvironment may both enhance understanding of pathogenesis and provide the framework for identification and validation of novel molecular targets.

To examine the effects of recombinant <em>growth</em> <em>factors</em> in vivo, impaired wound healing was studied in genetically diabetic C57BL/KsJ-db/db mice. Large full-thickness skin wounds made on the backs of these mice exhibited significant delays in the entry of inflammatory cells into the wound, the formation of granulation tissue, and in wound closure when compared to their nondiabetic littermates. Recombinant human platelet-derived <em>growth</em> <em>factor</em> (rPDGF-BB, 1 or 10 micrograms), recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (rbFGF, 1 micrograms), or combinations of both were applied topically to the wounds for 5 to 14 days after wounding. Diabetic mouse wounds treated with rPDGF-BB or rbFGF had many more <em>fibroblasts</em> and capillaries in the wound bed at 10 and <em>21</em> days than did wounds treated with the vehicle alone. The animals treated with <em>growth</em> <em>factors</em> also had significantly greater wound closure at <em>21</em> days than those treated with the vehicle. Combinations of rPDGF-BB and rbFGF improved all parameters of healing but not to a greater extent than either <em>growth</em> <em>factor</em> alone. The effectiveness of rPDGF-BB and rbFGF suggest that recombinant <em>growth</em> <em>factors</em> may be useful in the treatment of patients with deficient wound repair.

Starvation blocks the actions of <em>growth</em> hormone (GH) and inhibits <em>growth</em> through mechanisms that are not well understood. In this report, we demonstrate that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hormone induced by fasting, causes GH resistance. In liver, FGF<em>21</em> reduces concentrations of the active form of signal transducer and activator of transcription 5 (STAT5), a major mediator of GH actions, and causes corresponding decreases in the expression of its target genes, including insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1). FGF<em>21</em> also induces hepatic expression of IGF-1 binding protein 1 and suppressor of cytokine signaling 2, which blunt GH signaling. Chronic exposure to FGF<em>21</em> markedly inhibits <em>growth</em> in mice. These data suggest a central role for FGF<em>21</em> in inhibiting <em>growth</em> as part of its broader role in inducing the adaptive response to starvation.

Due to the poor prognosis of pancreatic cancer, novel diagnostic modalities for early diagnosis and new therapeutic strategy are urgently needed. Recently, microRNA-<em>21</em> (miR-<em>21</em>) was reported to be strongly overexpressed in pancreatic cancer as well as in other solid cancers. We investigated the functional roles of miR-<em>21</em>, which have not been fully elucidated in pancreatic cancer. miR-<em>21</em> expression was assessed in pancreatic cancer cell lines (14 cancer cell lines, primary cultures of normal pancreatic epithelial cells and <em>fibroblasts</em>, and a human normal pancreatic ductal epithelial cell line) and pancreatic tissue samples (25 cancer tissues and 25 normal tissues) by quantitative real-time reverse transcription-PCR amplification. Moreover, we investigated the proliferation, invasion, and chemoresistance of pancreatic cancer cells transfected with miR-<em>21</em> precursor or inhibitor. miR-<em>21</em> was markedly overexpressed in pancreatic cancer cells compared with nonmalignant cells, and miR-<em>21</em> in cancer tissues was much higher than in nonmalignant tissues. The cancer cells transfected with the miR-<em>21</em> precursor showed significantly increased proliferation, Matrigel invasion, and chemoresistance for gemcitabine compared with the control cells. In contrast, inhibition of miR-<em>21</em> decreased proliferation, Matrigel invasion, and chemoresistance for gemcitabine. Moreover, miR-<em>21</em> positively correlated with the mRNA expression of invasion-related genes, matrix metalloproteinase-2 and -9, and vascular endothelial <em>growth</em> <em>factor</em>. These data suggest that miR-<em>21</em> expression is increased in pancreatic cancer cells and that miR-<em>21</em> contributes to the cell proliferation, invasion, and chemoresistance of pancreatic cancer.

Scatter <em>factor</em> (SF), a secretory protein of <em>fibroblasts</em>, dissociates and increases the motility of epithelial cells and may be involved in cell migration processes during embryogenesis and tumor progression. Hepatocyte <em>growth</em> <em>factor</em> (HGF), a protein isolated from serum of patients with liver failure, is a potent mitogen for hepatocytes and is thought to play a role in liver regeneration. Here we present structural and functional evidence that human SF and human HGF (and also the human lung <em>fibroblast</em>-derived mitogen) are identical proteins encoded by a single gene, since (i) no major difference could be found by protein sequencing, by cDNA analysis, and by immunological comparison and (ii) SF in fact acts as a hepatocyte <em>growth</em> <em>factor</em>--i.e., stimulates DNA synthesis of activity--i.e., dissociates and induces invasiveness of various epithelial cells. The human SF/HGF gene was localized to chromosome bands 7q11.2-<em>21</em>. These results have important consequences for further studies on the involvement of SF/HGF as a modulator of cellular <em>growth</em> and motility in embryonal, malignant, and regenerative processes.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) is a recently discovered metabolic regulator. Here, we investigated the effects of FGF-<em>21</em> in the pancreatic beta-cell. In rat islets and INS-1E cells, FGF-<em>21</em> activated extracellular signal-regulated kinase 1/2 and Akt signaling pathways. In islets isolated from healthy rats, FGF-<em>21</em> increased insulin mRNA and protein levels but did not potentiate glucose-induced insulin secretion. Islets and INS-1E cells treated with FGF-<em>21</em> were partially protected from glucolipotoxicity and cytokine-induced apoptosis. In islets isolated from diabetic rodents, FGF-<em>21</em> treatment increased islet insulin content and glucose-induced insulin secretion. Short-term treatment of normal or db/db mice with FGF-<em>21</em> lowered plasma levels of insulin and improved glucose clearance compared with vehicle after oral glucose tolerance testing. Constant infusion of FGF-<em>21</em> for 8 weeks in db/db mice nearly normalized fed blood glucose levels and increased plasma insulin levels. Immunohistochemistry of pancreata from db/db mice showed a substantial increase in the intensity of insulin staining in islets from FGF-<em>21</em>-treated animals as well as a higher number of islets per pancreas section and of insulin-positive cells per islet compared with control. No effect of FGF-<em>21</em> was observed on islet cell proliferation. In conclusion, preservation of beta-cell function and survival by FGF-<em>21</em> may contribute to the beneficial effects of this protein on glucose homeostasis observed in diabetic animals.

Dysregulation of oncogenes by translocation to the IgH locus (14q32) is a seminal event in the pathogenesis of B-cell tumours. In multiple myeloma (MM), translocations to the IgH locus have been reported at an incidence of 20-60%. For most translocations, the partner chromosome is unknown (14q+); for the others, a diverse array of chromosomal partners have been identified, with 11q13 (cyclin D1) the only chromosome that is frequently involved. Recently, we developed a Southern-blot assay that detects translocation breakpoint fragments in most MM tumours, including those with no translocation detected by conventional karyotyping. In a continuing analysis of translocation in <em>21</em> myeloma cell lines and primary tumours, we show that the novel, karyotypically silent translocation t(4;14)(p16.3;q32.3) is present in five lines and at least three of ten primary tumours. The chromosome-4 breakpoints are clustered in a 70-kb region centromeric to the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 gene (FGFR3), the apparent dysregulated oncogene. Two lines and one primary tumour with this translocation selectively express an FGFR3 allele containing activating mutations identified previously in thanatophoric dwarfism. We propose that after the t(4;14) translocation, somatic mutation during tumour progression frequently generates in FGFR3 protein that is active in the absence of ligand.

<em>Fibroblast</em> <em>Growth</em> <em>Factors</em> (FGFs) are polypeptides with diverse activities in development and physiology. The mammalian Fgf family can be divided into the intracellular Fgf11/12/13/14 subfamily (iFGFs), the hormone-like Fgf15/<em>21</em>/23 subfamily (hFGFs), and the canonical Fgf subfamilies, including Fgf1/2/5, Fgf3/4/6, Fgf7/10/22, Fgf8/17/18, and Fgf9/16/20. However, all Fgfs are evolutionarily related. We propose that an Fgf13-like gene is the ancestor of the iFgf subfamily and the most likely evolutionary ancestor of the entire Fgf family. Potential ancestors of the canonical and hFgf subfamilies, Fgf4-, Fgf5-, Fgf8-, Fgf9-, Fgf10-, and Fgf15-like, appear to have derived from an Fgf13-like ancestral gene. Canonical FGFs function in a paracrine manner, while hFGFs function in an endocrine manner. We conclude that the ancestral Fgfs for these subfamilies acquired this functional diversity before the evolution of vertebrates. During the evolution of early vertebrates, the Fgf subfamilies further expanded to contain three or four members in each subfamily.

OBJECTIVE

Levels of parathyroid hormone (PTH) and the phosphaturic hormone FGF23, a fibroblast growth factor (FGF) family member, increase early in chronic kidney disease (CKD) before the occurrence of hyperphosphatemia. This short-term 6-wk dose titration study evaluated the effect of two phosphate binders on PTH and FGF23 levels in patients with CKD stages 3 to 4.

METHODS

Patients were randomized to receive over a 6-wk period either calcium acetate (n = 19) or sevelamer hydrochloride (n = 21).

RESULTS

At baseline, patients presented with elevated fractional excretion of phosphate, serum PTH, and FGF23. During treatment with both phosphate binders there was a progressive decline in serum PTH and urinary phosphate, but no change in serum calcium or serum phosphate. Significant changes were observed for FGF23 only in sevelamer-treated patients.

CONCLUSIONS

This study confirms the positive effects of early prescription of phosphate binders on PTH control. Prospective and long-term studies are necessary to confirm the effects of sevelamer on serum FGF23 and the benefits of this decrease on outcomes.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has been identified as a potent metabolic regulator. Administration of recombinant FGF<em>21</em> protein to rodents and rhesus monkeys with diet-induced or genetic obesity and diabetes exerts strong antihyperglycemic and triglyceride-lowering effects and reduction of body weight. Despite the importance of FGF<em>21</em> in the regulation of glucose, lipid, and energy homeostasis, the mechanisms by which FGF<em>21</em> functions as a metabolic regulator remain largely unknown. Here we demonstrate that FGF<em>21</em> regulates energy homeostasis in adipocytes through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), resulting in enhanced mitochondrial oxidative function. AMPK phosphorylation levels were increased by FGF<em>21</em> treatment in adipocytes as well as in white adipose tissue from ob/ob mice. FGF<em>21</em> treatment increased cellular NAD(+) levels, leading to activation of SIRT1 and deacetylation of its downstream targets, peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) and histone 3. Activation of AMPK and SIRT1 by FGF<em>21</em> in adipocytes enhanced mitochondrial oxidative capacity as demonstrated by increases in oxygen consumption, citrate synthase activity, and induction of key metabolic genes. The effects of FGF<em>21</em> on mitochondrial function require serine/threonine kinase 11 (STK11/LKB1), which activates AMPK. Inhibition of AMPK, SIRT1, and PGC-1alpha activities attenuated the effects of FGF<em>21</em> on oxygen consumption and gene expression, indicating that FGF<em>21</em> regulates mitochondrial activity and enhances oxidative capacity through an AMPK-SIRT1-PGC1alpha-dependent mechanism in adipocytes.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a key metabolic regulator. Expressed primarily in liver and adipose tissue, FGF<em>21</em> is induced via peroxisome proliferator-activated receptor (PPAR) pathways during states requiring increased fatty acid oxidation including fasting and consumption of a ketogenic diet. To test the hypothesis that FGF<em>21</em> is a physiological regulator that plays a role in lipid oxidation, we generated mice with targeted disruption of the Fgf<em>21</em> locus (FGF<em>21</em> knockout). Mice lacking FGF<em>21</em> had mild weight gain and slightly impaired glucose homeostasis, indicating a role in long-term energy homeostasis. Furthermore, FGF<em>21</em>KO mice tolerated a 24-h fast, indicating that FGF<em>21</em> is not essential in the early stages of starvation. In contrast to wild-type animals in which feeding KD leads to dramatic weight loss, FGF<em>21</em>KO mice fed KD gained weight, developed hepatosteatosis, and showed marked impairments in ketogenesis and glucose control. This confirms the physiological importance of FGF<em>21</em> in the adaptation to KD feeding. At a molecular level, these effects were accompanied by lower levels of expression of PGC1alpha and PGC1beta in FGF<em>21</em>KO mice, strongly implicating these key transcriptional regulators in the action of FGF<em>21</em>. Furthermore, within the liver, the maturation of the lipogenic transcription <em>factor</em> sterol regulatory element-binding protein-1c was increased in FGF<em>21</em>KO mice, implicating posttranscriptional events in the maladaptation of FGF<em>21</em>KO mice to KD. These data reinforce the role of FGF<em>21</em> is a critical regulator of long-term energy balance and metabolism. Mice lacking FGF<em>21</em> cannot respond appropriately to a ketogenic diet, resulting in an impaired ability to mobilize and utilize lipids.

Enhanced <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) production and circulation has been linked to the metabolic adaptation to starvation. Here, we demonstrated that hepatic FGF<em>21</em> expression is induced by dietary protein restriction, but not energy restriction. Circulating FGF<em>21</em> was increased 10-fold in mice and rats fed a low-protein (LP) diet. In these animals, liver Fgf<em>21</em> expression was increased within 24 hours of reduced protein intake. In humans, circulating FGF<em>21</em> levels increased dramatically following 28 days on a LP diet. LP-induced increases in FGF<em>21</em> were associated with increased phosphorylation of eukaryotic initiation <em>factor</em> 2α (eIF2α) in the liver, and both baseline and LP-induced serum FGF<em>21</em> levels were reduced in mice lacking the eIF2α kinase general control nonderepressible 2 (GCN2). Finally, while protein restriction altered food intake, energy expenditure, and body weight gain in WT mice, FGF<em>21</em>-deficient animals did not exhibit these changes in response to a LP diet. These and other data demonstrate that reduced protein intake underlies the increase in circulating FGF<em>21</em> in response to starvation and a ketogenic diet and that FGF<em>21</em> is required for behavioral and metabolic responses to protein restriction. FGF<em>21</em> therefore represents an endocrine signal of protein restriction, which acts to coordinate metabolism and <em>growth</em> during periods of reduced protein intake.

Oncogenic osteomalacia (OO) is a rare paraneoplastic syndrome of osteomalacia due to phosphate wasting. The phosphaturic mesenchymal tumor (mixed connective tissue variant) (PMTMCT) is an extremely rare, distinctive tumor that is frequently associated with OO. Despite its association with OO, many PMTMCTs go unrecognized because they are erroneously diagnosed as other mesenchymal tumors. Expression of <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23), a recently described protein putatively implicated in renal tubular phosphate loss, has been shown in a small number of mesenchymal tumors with known OO. The clinicopathological features of 32 mesenchymal tumors either with known OO (29) or with features suggestive of PMTMCT (3) were studied. Immunohistochemistry for cytokeratin, S-100, actin, desmin, CD34, and FGF-23 was performed. The patients (13 male, 19 female) ranged from 9 to 80 years in age (median 53 years). A long history of OO was common. The cases had been originally diagnosed as PMTMCT (15), hemangiopericytoma (HPC) (3), osteosarcoma (3), giant cell tumor (2), and other (9). The tumors occurred in a variety of soft tissue (<em>21</em>) and bone sites (11) and ranged from 1.7 to 14 cm. Twenty-four cases were classic PMTMCT with low cellularity, myxoid change, bland spindled cells, distinctive "grungy" calcified matrix, fat, HPC-like vessels, microcysts, hemorrhage, osteoclasts, and an incomplete rim of membranous ossification. Four of these benign-appearing PMTMCTs contained osteoid-like matrix. Three other PMTMCTs were hypercellular and cytologically atypical and were considered malignant. The 3 cases without known OO were histologically identical to the typical PMTMCT. Four cases did not resemble PMTMCT: 2 sinonasal HPC, 1 conventional HPC, and 1 sclerosing osteosarcoma. Three cases expressed actin; all other markers were negative. Expression of FGF-23 was seen in 17 of <em>21</em> cases by immunohistochemistry and in 2 of 2 cases by RT-PCR. Follow-up (25 cases, 6-348 months) indicated the following: <em>21</em> alive with no evidence of disease and with normal serum chemistry, 4 alive with disease (1 malignant PMTMCT with lung metastases). We conclude that most cases of mesenchymal tumor-associated OO, both in the present series and in the reported literature, are due to PMTMCT. Improved recognition of their histologic spectrum, including the presence of bone or osteoid-like matrix in otherwise typical cases and the existence of malignant forms, should allow distinction from other mesenchymal tumors. Recognition of PMTMCT is critical, as complete resection cures intractable OO. Immunohistochemistry and RT-PCR for FGF-23 confirm the role of this protein in PMTMCT-associated OO.

The metabolic regulator <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has antidiabetic properties in animal models of diabetes and obesity. Using quantitative RT-PCR, we here show that the hepatic gene expression of FGF<em>21</em> is regulated by the peroxisome proliferator-activated receptor alpha (PPARalpha). Fasting or treatment of mice with the PPARalpha agonist Wy-14,643 induced FGF<em>21</em> mRNA by 10-fold and 8-fold, respectively. In contrast, FGF<em>21</em> mRNA was low in PPARalpha deficient mice, and fasting or treatment with Wy-14,643 did not induce FGF<em>21</em>. Obese ob/ob mice, known to have increased PPARalpha levels, displayed 12-fold increased hepatic FGF<em>21</em> mRNA levels. The potential importance of PPARalpha for FGF<em>21</em> expression also in human liver was shown by Wy-14,643 induction of FGF<em>21</em> mRNA in human primary hepatocytes, and PPARalpha response elements were identified in both the human and mouse FGF<em>21</em> promoters. Further studies on the mechanisms of regulation of FGF<em>21</em> by PPARalpha in humans will be of great interest.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hepatokine that acts as a global starvation signal to modulate fuel partitioning and metabolism and repress <em>growth</em>; however, the site of action of these diverse effects remains unclear. FGF<em>21</em> signals through a heteromeric cell-surface receptor composed of one of three FGF receptors (FGFR1c, FGFR2c or FGFR3c) in complex with β-Klotho, a single-pass transmembrane protein that is enriched in metabolic tissues. Here we show that in addition to its known effects on peripheral metabolism, FGF<em>21</em> increases systemic glucocorticoid levels, suppresses physical activity and alters circadian behavior, which are all features of the adaptive starvation response. These effects are mediated through β-Klotho expression in the suprachiasmatic nucleus of the hypothalamus and the dorsal vagal complex of the hindbrain. Mice lacking the gene encoding β-Klotho (Klb) in these regions are refractory to these effects, as well as those on metabolism, insulin and <em>growth</em>. These findings demonstrate a crucial role for the nervous system in mediating the diverse physiologic and pharmacologic actions of FGF<em>21</em>.

The endocrine hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a powerful modulator of glucose and lipid metabolism and a promising drug for type 2 diabetes. Here we identify FGF<em>21</em> as a potent regulator of skeletal homeostasis. Both genetic and pharmacologic FGF<em>21</em> gain of function lead to a striking decrease in bone mass. In contrast, FGF<em>21</em> loss of function leads to a reciprocal high-bone-mass phenotype. Mechanistically, FGF<em>21</em> inhibits osteoblastogenesis and stimulates adipogenesis from bone marrow mesenchymal stem cells by potentiating the activity of peroxisome proliferator-activated receptor γ (PPAR-γ). Consequently, FGF<em>21</em> deletion prevents the deleterious bone loss side effect of the PPAR-γ agonist rosiglitazone. Therefore, FGF<em>21</em> is a critical rheostat for bone turnover and a key integrator of bone and energy metabolism. These results reveal that skeletal fragility may be an undesirable consequence of chronic FGF<em>21</em> administration.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> is an endocrine hormone that is expressed in multiple tissues and functions physiologically to maintain energy homeostasis. FGF<em>21</em> is being pursued as a therapeutic target for diabetes and obesity because of its rapid and potent effects on improving insulin sensitivity. However, whether FGF<em>21</em> enhances insulin sensitivity under physiologic conditions remains unclear. Here, we show that liver-derived FGF<em>21</em> enters the circulation during fasting but also remains present and functional during the early stage of refeeding. After a prolonged fast, FGF<em>21</em> acts as an insulin sensitizer to overcome the peripheral insulin resistance induced by fasting, thereby maximizing glucose uptake. Likewise, FGF<em>21</em> is produced from the liver during overfeeding and mitigates peripheral insulin resistance. DIO FGF<em>21</em> liver-specific knockout, but not FGF<em>21</em> adipose-specific knockout, mice have increased insulin resistance and decreased brown adipose tissue-mediated glucose disposal. These data are compatible with the concept that FGF<em>21</em> functions physiologically as an insulin sensitizer under conditions of acute refeeding and overfeeding.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a fasting-induced hepatokine that has potent pharmacologic effects in mice, which include improving insulin sensitivity and blunting <em>growth</em>. The single-transmembrane protein βKlotho functions as a coreceptor for FGF<em>21</em> in vitro. To determine if βKlotho is required for FGF<em>21</em> action in vivo, we generated whole-body and adipose tissue-selective βKlotho-knockout mice. All of the effects of FGF<em>21</em> on <em>growth</em> and metabolism were lost in whole-body βKlotho-knockout mice. Selective elimination of βKlotho in adipose tissue blocked the acute insulin-sensitizing effects of FGF<em>21</em>. Taken together, these data demonstrate that βKlotho is essential for FGF<em>21</em> activity and that βKlotho in adipose tissue contributes to the beneficial metabolic actions of FGF<em>21</em>.

A rat model of bleomycin-induced pulmonary inflammation and fibrosis was used to examine the relationship between collagen synthesis and transforming <em>growth</em> <em>factor</em> beta (TGF-beta) production, and cellular distribution. Total lung TGF-beta was elevated within 2 h of intratracheal bleomycin administration and peaked 7 d later at levels 30-fold higher than controls. This was followed by a gradual decline with lower but persistent levels of production in the late phase of the response between <em>21</em> and 28 d later. The peak TGF-beta levels preceded the maximum collagen and noncollagen protein synthesis measured by [3H]proline incorporation into lung <em>fibroblast</em> explants of bleomycin-treated rats. The pattern of immunohistochemical staining localized TGF-beta initially in the cytoplasm of bronchiolar epithelium cells and subepithelial extracellular matrix. The peak of lung TGF-beta levels at 7 d coincided with intense TGF-beta staining of macrophages dispersed in the alveolar interstitium and in organized clusters. Later in the course of the response. TGF-beta was primarily associated with extracellular matrix in regions of increased cellularity and tissue repair, and coincided with the maximum <em>fibroblast</em> collagen synthesis. This temporal and spatial relationship between collagen production and TGF-beta production by macrophages suggests an important if not primary role for TGF-beta in the pathogenesis of the pulmonary fibrosis.

<em>Growth</em> <em>factors</em>, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages. We demonstrate that mechanical stimulation in vitro, without ligament-selective exogenous <em>growth</em> and differentiation <em>factors</em>, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages). A bioreactor was designed to permit the controlled application of ligament-like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel. The application of mechanical stress over a period of <em>21</em> days up-regulated ligament <em>fibroblast</em> markers, including collagen types I and III and tenascin-C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells. At the same time, no up-regulation of bone or cartilage-specific cell markers was observed.