The ligand for osteoprotegerin (OPGL) directly activates mature osteoclasts.

Osteoprotegerin (OPG) and OPG-ligand (OPGL) potently inhibit and stimulate, respectively, osteoclast differentiation (Simonet, W.S., D.L. Lacey, C.R. Dunstan, M. Kelley, M.-S. Chang, R. Luethy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, et al. 1997. Cell. 89:309-319; Lacey, D.L., E. Timms, H.-L. Tan, M.J. Kelley, C.R. Dunstan, T. Burgess, R. Elliott, A. Colombero, G. Elliott, S. Scully, et al. 1998. Cell. 93: 165-176), but their effects on mature osteoclasts are not well understood. Using primary cultures of rat osteoclasts on bone slices, we find that OPGL causes approximately sevenfold increase in total bone surface erosion. By scanning electron microscopy, OPGL-treated osteoclasts generate more clusters of lacunae on bone suggesting that multiple, spatially associated cycles of resorption have occurred. However, the size of individual resorption events are unchanged by OPGL treatment. Mechanistically, OPGL binds specifically to mature OCs and rapidly (within 30 min) induces actin ring formation; a marked cytoskeletal rearrangement that necessarily precedes bone resorption. Furthermore, we show that antibodies raised against the OPGL receptor, RANK, also induce actin ring formation. OPGL-treated mice exhibit increases in blood ionized Ca++ within 1 h after injections, consistent with immediate OC activation in vivo. Finally, we find that OPG blocks OPGL's effects on both actin ring formation and bone resorption. Together, these findings indicate that, in addition to their effects on OC precursors, OPGL and OPG have profound and direct effects on mature OCs and indicate that the OC receptor, RANK, mediates OPGL's effects.

Neu differentiation factor upregulates epidermal migration and integrin expression in excisional wounds.

Neu differentiation factor (NDF) is a 44-kD glycoprotein which was isolated from ras-transformed rat fibroblasts and indirectly induces tyrosine phosphorylation of the HER-2/neu receptor via binding to either the HER-3 or HER-4 receptor. NDF contains a receptor binding epidermal growth factor (EGF)-like domain and is a member of the EGF family. There are multiple different isoforms of NDF which arise by alternative splicing of a single gene. To date, in vivo biologic activities have not been demonstrated for any NDF isoform. Since NDF, HER-2/neu, and HER-3 are present in skin, and other EGF family members can influence wound keratinocytes in vivo, we investigated whether NDF would stimulate epidermal migration and proliferation in a rabbit ear model of excisional wound repair. In this model, recombinant human NDF-alpha 2 (rhNDF-alpha 2), applied once at the time of wounding, induced a highly significant increase in both epidermal migration and epidermal thickness at doses ranging from 4 to 40 micrograms/cm2. In contrast, rhNDF-alpha 1, rhNDF-beta 1, and rhNDF-beta 2 had no apparent biologic effects in this model. rhNDF-alpha 2 also induced increased neoepidermal expression of alpha 5 and alpha 6 integrins, two of the earliest integrins to appear during epidermal migration. In addition, rhNDF-alpha 2-treated wounds exhibited increased neoepidermal expression of cytokeratin 10 and filaggrin, both epidermal differentiation markers. NDF alpha isoforms were expressed in dermal fibroblasts of wounded and unwounded skin, while both HER-2/neu and HER-3 were expressed in unwounded epidermis and dermal adnexa. In wounds, HER-2/neu expression was markedly decreased in the wound neoepidermis while neoepidermal HER-3 expression was markedly upregulated. Taken together, these results suggest that endogenous NDF-alpha 2 may function as a paracrine mediator directing initial epidermal migration during cutaneous tissue repair.

FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation.

The fibroblast growth factors (FGFs) play key roles in controlling tissue growth, morphogenesis, and repair in animals. We have cloned a novel member of the FGF family, designated FGF-18, that is expressed primarily in the lungs and kidneys and at lower levels in the heart, testes, spleen, skeletal muscle, and brain. Sequence comparison indicates that FGF-18 is highly conserved between humans and mice and is most homologous to FGF-8 among the FGF family members. FGF-18 has a typical signal sequence and was glycosylated and secreted when it was transfected into 293-EBNA cells. Recombinant murine FGF-18 protein (rMuFGF-18) stimulated proliferation in the fibroblast cell line NIH 3T3 in vitro in a heparan sulfate-dependent manner. To examine its biological activity in vivo, rMuFGF-18 was injected into normal mice and ectopically overexpressed in transgenic mice by using a liver-specific promoter. Injection of rMuFGF-18 induced proliferation in a wide variety of tissues, including tissues of both epithelial and mesenchymal origin. The two tissues which appeared to be the primary targets of FGF-18 were the liver and small intestine, both of which exhibited histologic evidence of proliferation and showed significant gains in organ weight following 7 (sometimes 3) days of FGF-18 treatment. Transgenic mice that overexpressed FGF-18 in the liver also exhibited an increase in liver weight and hepatocellular proliferation. These results suggest that FGF-18 is a pleiotropic growth factor that stimulates proliferation in a number of tissues, most notably the liver and small intestine.

Systemically and topically administered leptin both accelerate wound healing in diabetic ob/ob mice.

Leptin is a 16 kD protein that is produced by adipocytes and induces weight loss in both normal and genetically obese ob/ob mice. ob/ob mice are obese, have multiple metabolic abnormalities, and exhibit impaired wound healing. Exogenous administration of leptin to these animals induces weight loss and corrects their metabolic defects. Leptin's effect on wound repair, however, has not been studied. Systemic administration of leptin at doses ranging from 0.1 to 10 mg/kg/day induced a highly significant acceleration in wound repair in ob/ob mice (p<0.0001), but not in db/db mice, indicating that leptin's effects on wound repair were mediated through the leptin receptor. We then investigated the possibility that leptin was acting directly at the wound site by administering leptin topically, and found that topical leptin also induced a dose dependent acceleration in wound repair (p<0.0001). In addition, we found that all forms of leptin receptor, including the signal transducing long form, were present in skin by RNase protection assay, and that leptin receptor localized to subcutaneous vessels of wounded skin by in situ hybridization. Finally, we investigated the possibility that leptin stimulated angiogenesis in wounds by analyzing wound hemoglobin and wound vessel density. Neither systemic nor topical leptin induced any significant changes in either parameter, suggesting that leptin accelerates wound repair by a mechanism other than stimulation of angiogenesis. In summary, both systemic and topical leptin accelerate wound repair in diabetic ob/ob mice, possibly via the direct interaction of leptin with its receptors in wounded skin, but do not appear to significantly stimulate wound angiogenesis. Further studies to better elucidate the mechanisms of leptin's effects on wound repair are warranted.

Growth factors and cytokines in hair follicle development and cycling: recent insights from animal models and the potentials for clinical therapy.

Hair growth disorders, particularly those that lead to hair loss (alopecia), are common and frequently cause significant mental anguish in affected individuals. The mechanisms underlying the majority of these disorders are unknown. However, insights into the specific molecular mechanisms of hair follicle development and cycling have recently been made using animal models, particularly mice that over- or underexpress a specific gene for a growth factor or cytokine. Other animal models have demonstrated that certain growth factors and cytokines can prevent much of the alopecia caused by cancer chemotherapeutic agents. These animal models have confirmed the importance of growth factors and cytokines in hair follicle development and cycling, and have formed the foundation for potential clinical therapy of hair growth disorders, particularly alopecia. Nevertheless, important questions concerning their efficacy, safety and delivery will need to be answered before successful clinical therapy of any hair growth disorder becomes a reality.

Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Normalization of the nu/nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia.

The growth and development of hair follicles is influenced by a number of different growth factors and cytokines, particularly members of the fibroblast growth factor (FGF) family. Keratinocyte growth factor (KGF or FGF-7) is a recently identified 28-kd member of the FGF family that induces proliferation of a wide variety of epithelial cells, including keratinocytes within the epidermis and dermal adnexa. Because KGF induces marked proliferation of keratinocytes, and both KGF and KGF receptor (KGFR) mRNA are expressed at high levels in skin, we sought to localize KGF and KGFR in skin by in situ hybridization. KGFR mRNA was relatively strongly expressed by keratinocytes in the basilar epidermis as well as throughout developing hair follicles of rat embryos and neonates. KGF mRNA was expressed at lower levels than was KGFR but could be localized to follicular dermal papillae in rat embryos and neonates. These results prompted us to investigate the effects of KGF on hair follicles in two distinct murine models of alopecia. In the first model, recombinant KGF (rKGF) induced dose-dependent hair growth over most of the body in nu/nu athymic nude mice when administered intraperitoneally or subcutaneously over 17 to 18 days. When administered subcutaneously, rKGF induced the most extensive hair growth at the sites of injection. Histologically, rKGF induced marked follicular and sebaceous gland hypertrophy, a normalization of the nu/nu follicular keratinization defect, and an increase in follicular keratinocyte proliferation as assessed by bromodeoxyuridine labeling. In the second model, a neonatal rat model of cytosine arabinoside chemotherapy-induced alopecia in which interleukin-1, epidermal growth factor, and acidic FGF have all demonstrated some degree of alopecia cytoprotection, rKGF induced a dose-dependent cytoprotective effect, abrogating as much as 50% of the alopecia in this model when administered beginning 1 day before the onset of chemotherapy. Taken together, these data suggest that KGF is an important endogenous mediator of normal hair follicle growth, development, and differentiation.

Growth factors in porcine full and partial thickness burn repair. Differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor, and neu differentiation factor.

The topical application of recombinant growth factors such as epidermal growth factor, platelet-derived growth factor-BB homodimer (rPDGF-BB), keratinocyte growth factor (rKGF), and neu differentiation factor has resulted in significant acceleration of healing in several animal models of wound repair. In this study, we established highly reproducible and quantifiable full and deep partial thickness porcine burn models in which burns were escharectomized 4 or 5 days postburn and covered with an occlusive dressing to replicate the standard treatment in human burn patients. We then applied these growth factors to assess their efficacy on several parameters of wound repair: extracellular matrix and granulation tissue production, percent reepithelialization, and new epithelial area. In full thickness burns, only rPDGF-BB and the combination of rPDGF-BB and rKGF induced significant changes in burn repair. rPDGF-BB induced marked extracellular matrix and granulation tissue production (P = 0.013) such that the burn defect was filled within several days of escharectomy, but had no effect on new epithelial area or reepithelialization. The combination of rPDGF-BB and rKGF in full thickness burns resulted in a highly significant increase in extracellular matrix and granulation tissue area (P = 0.0009) and a significant increase in new epithelial area (P = 0.007), but had no effect on reepithelialization. In deep partial thickness burns, rKGF induced the most consistent changes. Daily application of rKGF induced a highly significant increase in new epithelial area (P < 0.0001) but induced only a modest increase in reepithelialization (83.7% rKGF-treated versus 70.2% control; P = 0.016) 12 days postburn. rKGF also doubled the number of fully reepithelialized burns (P = 0.02) at 13 days postburn, at least partially because of marked stimulation of both epidermal and follicular proliferation as assessed by proliferating cell nuclear antigen expression. In situ hybridization for KGFR in porcine burns revealed strong expression of KGFR on hair follicles and basal epidermis, confirming direct rKGF action on follicular as well as epidermal keratinocytes. Although the epithelial proliferation induced by rKGF resulted in marked neoepidermal psoriasiform hyperplasia with exaggerated rete ridges and neoepidermal and follicular maturation as assessed by expression of cytokeratin 10, a marker of keratinocyte terminal differentiation was not delayed and appeared to be accelerated in some rKGF-treated burns. Recombinant epidermal growth factor induced a trend toward increased new epithelial area in deep partial thickness burns, but had no effect on reepithelialization. The recombinant neu differentiation factor-alpha 2 isoform had no significant biological effects in either full or deep partial thickness burns.(ABSTRACT TRUNCATED AT 250 WORDS)

Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless.

Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10(-/-) fetuses continued to develop until birth, despite the complete absence of both fore- and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10(-/-) mice exhibited perinatal lethality associated with complete absence of lungs. Although tracheal development was normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, was completely disrupted. The pulmonary phenotype of Fgf-10(-/-) mice is strikingly similar to that of the Drosophila mutant branchless, an Fgf homolog.