Identification of a novel fibroblast growth factor, FGF-23, preferentially expressed in the ventrolateral thalamic nucleus of the brain

We isolated mouse cDNA encoding a novel FGF (251 amino acids). As this is the 23rd documented FGF, we termed it FGF-23. FGF-23 has a hydrophobic amino terminus ( approximately 24 amino acids), which is a typical signal sequence. As expected, recombinant mouse FGF-23 was efficiently secreted by High Five insect cell-infected recombinant baculovirus containing the cDNA, indicating that FGF-23 is a secreted protein. We also isolated human cDNA encoding FGF-23 (251 amino acids), which is highly identical ( approximately 72% amino acid identity) to mouse FGF-23. Of human FGF family members, FGF-23 is most similar to FGF-21 and FGF-19 ( approximately 24% and approximately 22% amino acid identities, respectively). Human FGF-23 gene was localized on the chromosome 12p13 and found to be tandem linked (within 5.5 kb) to human FGF-6 gene. The expression of FGF-23 mRNA in mouse adult tissues was examined by real-time quantitative polymerase chain reaction. FGF-23 mRNA was mainly expressed in the brain and thymus at low levels. The localization of FGF-23 mRNA in the brain was examined by in situ hybridization. FGF-23 mRNA in the brain was found to be preferentially expressed in the ventrolateral thalamic nucleus. Therefore, FGF-23 is expected a unique FGF that plays roles in the function of the ventrolateral thalamic nucleus.     

Identification of a novel FGF, FGF-21, preferentially expressed in the liver

We isolated cDNA encoding a novel FGF (210 amino acids) from mouse embryos. As this is the 21st documented FGF, we tentatively term it FGF-21. FGF-21 has a hydrophobic amino terminus ( approximately 30 amino acids), which is a typical signal sequence, and appears to be a secreted protein. The expression of FGF-21 mRNA in mouse adult tissues was examined by Northern blotting analysis. FGF-21 mRNA was most abundantly expressed in the liver, and also expressed in the thymus at lower levels. We also isolated human cDNA encoding FGF-21 (209 amino acids). Human FGF-21 is highly identical ( approximately 75% amino acid identity) to mouse FGF-21. Among human FGF family members, FGF-21 is most similar ( approximately 35% amino acid identity) to FGF-19.     

Cloning and characterization of a cDNA encoding a novel fibroblast growth factor preferentially expressed in human heart

A novel human fibroblast growth factor (hFGF), which shows 75% sequence homology with fibroblast growth factor-9, was isolated in random sequencing of a human heart cDNA library. The full-length sequence is 928 bp, the encoded protein is composed of 168 amino acid residues, and its pI value and molecular weight were estimated to be 8.13 and 19.1 kDa, respectively. RT-PCR using Marathon human heart cDNA shows that the coding region is approximately 507 bp. Southern hybridization showed a single band which indicates that this is a single copy gene. Northern hybridization done on a human multiple tissues blot showed that the gene is preferentially expressed in human heart, very weakly detectable in human brain and not detectable in 18 other different human tissues.     

A novel fibroblast growth factor receptor-5 preferentially expressed in the pancreas(1)

Using the polymerase chain reaction on human embryonic cDNAs, we isolated a cDNA encoding a novel 504 amino acid protein, termed fibroblast growth factor receptor (FGFR)-5, which is highly homologous to known FGFRs. The NH(2)-terminal portion of FGFR5 contains a putative secretory signal sequence, three typical immunoglobulin-like domains, six cysteines, and an acidic box, but no HAV motif. The COOH-terminal portion of FGFR5 contains one transmembrane domain but no intracellular kinase domain. Recombinant FGFR5 expressed in COS-7 cells is not secreted, but recombinant truncated FGFR5 lacking the predicted transmembrane domain is secreted. Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) do not bind to FGFR5. Among 23 adult human tissues, FGFR5 mRNA is preferentially expressed in the pancreas. These results suggest that FGFR5 may provide a binding site for some other fibroblast growth factors and may regulate some pancreatic function.     

Characterization of FGFRL1, a novel fibroblast growth factor (FGF) receptor preferentially expressed in skeletal tissues

Clones for a novel transmembrane receptor termed FGFRL1 were isolated from a subtracted, cartilage-specific cDNA library prepared from chicken sterna. Homologous sequences were identified in other vertebrates, including man, mouse, rat and fish, but not in invertebrates such as Caenorhabditis elegans and Drosophila. FGFRL1 was expressed preferentially in skeletal tissues as demonstrated by Northern blotting and in situ hybridization. Small amounts of the FGFRL1 mRNA were also detected in other tissues such as skeletal muscle and heart. The novel protein contained three extracellular Ig-like domains that were related to the members of the fibroblast growth factor (FGF) receptor family. However, it lacked the intracellular protein tyrosine kinase domain required for signal transduction by transphosphorylation. When expressed in cultured cells as a fusion protein with green fluorescent protein, FGFRL1 was specifically localized to the plasma membrane where it might interact with FGF ligands. Recombinant FGFRL1 protein was produced in a baculovirus system with intact disulfide bonds. Similar to FGF receptors, the expressed protein interacted specifically with heparin and with FGF2. When overexpressed in MG-63 osteosarcoma cells, the novel receptor had a negative effect on cell proliferation. Taken together our data are consistent with the view that FGFRL1 acts as a decoy receptor for FGF ligands.     

FGF-20, a novel neurotrophic factor, preferentially expressed in the substantia nigra pars compacta of rat brain

We have isolated cDNA encoding a novel FGF (212 amino acids) from rat brain. Because this is the 20th documented member of the FGF family, we tentatively term it FGF-20. Among FGF family members, FGF-20 is most similar to FGF-9 and FGF-16 (70 and 62% amino acid identity, respectively). Human FGF-20 gene was found in the human genomic sequence mapped to the 8p21.3-p22 region. Human FGF-20 is highly identical to rat FGF-20 (95% amino acid identity). FGF-20 mRNA was preferentially expressed in rat brain among the adult major tissues examined. The localization of FGF-20 mRNA in rat brain was also examined by in situ hybridization. FGF-20 mRNA was preferentially expressed in the substantia nigra pars compacta. To examine the biological activity of FGF-20, recombinant rat FGF-20 was produced by insect cells infected with recombinant baculovirus containing rat FGF-20 cDNA. Recombinant rat FGF-20 enhanced the survival of midbrain dopaminergic neurons. The present results indicate that FGF-20 is a novel neurotrophic factor preferentially expressed in the substantia nigra pars compacta of rat brain.     

Identification of genes preferentially expressed in goat hair follicle anagen-catagen transition using suppression subtractive hybridization

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes in goat (Capra hircus) hair follicle anagen-catagen transition. The cDNA fragments, derived from SSH positive subtractive library (tester: anagen-catagen transition, driver: later anagen), were cloned into pEGM-T vector. Two hundred cDNA fragments screened from this library were subjected to identify forty-five unregulated isolates. Sequence analysis revealed that these fragments represented twenty-three genes. Blasting analysis with database in GenBank showed that twenty genes were previously clearly annotated, two were homologous to un-annotated expressed sequence tag (ESTs), and one might be novel. To identify characters of gene expression, seven genes in later anagen and anagen-catagen transition skin tissues were chosen for quantitative real-time PCR. Results indicated that expression of these seven genes varied much, reaching threefold among them, furthering indicating that expression of those genes was up-regulation in the anagen-catagen transition. We characterized expression levels of this potential novel gene and the goat ectodysplasin A during differential stages of hair cycle. These profiles suggested that these two genes might play a role in the goat secondary hair follicle cycle.     

The electron microscopy of the human hair follicle. III. The inner root sheath and trichohyaline

1. The three cylinders of cells, each one cell thick, which together constitute the inner root sheath, arise from the peripheral portions of the undifferentiated matrix. These cells, like the hair cuticle, are stabilised by the spread of adhesive contacts between their plasma membranes which occurs in the mid-bulb and upper bulb of the hair follicle. 2. The characteristic intracellular product of all three cell layers is trichohyaline. This substance is formed in the first place as amorphous droplets which subsequently transform into a birefringent form. 3. This transformation, involving the formation of a birefringent product from an amorphous precursor, is in contrast to the formation in the cortex of keratin which originates in a fibrous form. 4. Trichohyaline appears first and transforms first in the cells of Henle which are nearest the outer sheath and the dermal supply vessels. This transformation occurs at the level of the neck of the follicle. Synthesis and transformation in the cells of Huxley and the sheath cuticle lag behind the similar events in the cells of Henle. The transformation does not begin until the lower prekeratinous zone in the Huxley and cuticle cells. 5. The amorpous-fibrous transformation occurs rapidly cell by cell and involves the conversion of all the trichohyaline droplets. In longitudinal sections the birefringent modification can be seen extending from the droplets in both directions parallel to the axis of the hair. In cross-sections the images of the transformed material are difficult to interpret. They may be seen as sections of corrugated sheets ( approximately 100 A thick) or condensed fibrils approximately 100 A in width. 6. At the same time that the trichohyaline transforms, the spacing between the cell membranes increases and a dark deposit appears centrally between them. This membrane complex, and the similar complex of the hair cuticle cells described in Part 2, may be specialised formations whose purpose is to hold the hardened cells together.     

Cellular interrelationships of the outer root sheath of the hair follicle

The 3H-thymidine labelling has demonstrated that into the internal (accompanying) layer of the outer root sheath (ORS) of the hain follicle the cells incorporate not only in the bulb but above it from the external layers of the ORS. Polymorphism of the ORS cells has been revealed electron microscopically; it can be interpreted as a result of transfer of some motionless, greatly vacuolized, containing a lot of glycogen cells to the cells with electron opaque cytoplasm, radially elongated and moving deep into up to their contact with the internal root sheath. The presence in the external layer of the ORS, at the level of the bulb and above it, some nonlabelled cells even 3 days after 3H-thymidine administration demonstrates against their origin from the cambial cells.     

FGF-19, a novel fibroblast growth factor with unique specificity for FGFR4.

We have identified a novel fibroblast growth factor, FGF-19, the most distant member of the FGF family described to date. FGF-19 is a high affinity, heparin dependent ligand for FGFR4 and is the first member of the FGF family to show exclusive binding to FGFR4. Human FGF-19 maps to chromosome 11 q13.1, a region associated with an osteoporosis-pseudoglioma syndrome of skeletal and retinal defects. FGF-19 message is expressed in several tissues including fetal cartilage, skin, and retina, as well as adult gall bladder and is overexpressed in a colon adenocarcinoma cell line.     

Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.     

Keratins of the human hair follicle: "Hyperproliferative''keratins consistently expressed in outer root sheath cells in vivo and in vitro

Keratins produced by morphologically distinct compartments of the human hair folicle (hHF) were analysed and compared to those produced by cultured hHF and interfollicular keratinocytes. Five of the major keratins, the basic keratins nos. 5 and 6 (apparent mol. mass 60 and 58 kDa) and the acidic keratins nos. 14, 16, and 17 (51, 49 and 48 kDa), could be labelled in intact hHF and were found in all fractions of the outer root sheath (ORS). The other major keratins, which were not labelled under these conditions (basic-neutral hHbI and -II; 60-62 kDa and acidic hHaI and -II; 40-42 kDa) were associated with hair shaft (hHS) both in the follicle and, virtually unchanged, in the distal part of the hair. Another, previously undescribed, group of proteins with keratin-like properties exhibiting a broad pI-spectrum (basic to slightly acidic: hIC-I, -II, -III, 64-67 kDa; distinctly acidic: hIC-IV, about 54 kDa) was detected in isolated inner root sheath (IRS), in the cuticular material shed from denuded hHS, and also in nail plates. In our experiments only ORS cells grew readily in culture irrespective of their origin from peripheral (mesenchyme-adjacent) or more central ORS-cell layers. In contrast to keratinocytes from interfollicular epidermis (IFE) the cultured ORS cells expressed a keratin set virtually identical to that expressed in vivo. This set also closely resembled that expressed by IFE keratinocyte cultures. The identity of the respective keratins (nos. 5, 6, 14, 16, and 17) present in all these cells in vivo and in vitro was confirmed by tryptic peptide mapping. The data indicated that the microenvironment (in situ) directs the differentiation of ORS cells in a manner comparable to the way it is directed by conventional culture conditions, with consistent expression of the "basal" and "hyperproliferative" set of keratins. This, however, does not exclude the possibility that other types of environmentally induced response may occur, as seen for example during the reepithelialization of superficial skin wounds by ORS cells.     

Synthesis of porcine pCLCA2 protein during late differentiation of keratinocytes of epidermis and hair follicle inner root sheath

Despite the discovery of the widely expressed CLCA (chloride channel regulators, calcium-activated) proteins more than 15?years ago, their seemingly diverse functions are still poorly understood. With the recent generation of porcine animal models for cystic fibrosis (CF), members of the porcine CLCA family are becoming of interest as possible modulators of the disease in the pig. Here, we characterize pCLCA2, the porcine ortholog of the human hCLCA2 and the murine mCLCA5, which are the only CLCA members expressed in the skin. Immunohistochemical studies with a specific antibody against pCLCA2 have revealed a highly restricted pCLCA2 protein expression in the skin. The protein is strictly co-localized with filaggrin and trichohyalin in the granular layer of the epidermis and the inner root sheath of the hair follicles, respectively. No differences have been observed between the expression patterns of wild-type pigs and CF transmembrane conductance regulator ^-/- pigs. We speculate that pCLCA2 plays an as yet undefined role in the structural integrity of the skin or, possibly, in specialized functions of the epidermis, including barrier or defense mechanisms.     

Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle.

Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10-derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization.     

Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.     

Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5

Earlier studies demonstrated that knock-out of fibroblast growth factor-5 gene (Fgf-5) prolonged anagen VI phase of hair cycle, resulting long hairs in the mice. We showed the activities on hair growth of the two Fgf-5 gene products, one of which, FGF-5 suppressed hair growth by inhibiting anagen proceeding and inducing the transition from anagen to catagen, and FGF-5S, a shorter polypeptide with FGF-5-antagonizing activity translated from alternatively spliced mRNA, suppressed this activity of FGF-5. As the results suggested that FGF-5 antagonist would increase hair growth, we synthesized various peptides having partial sequences of human FGF-5 and FGF-5S and determined their FGF-5 antagonist activity. Among them, a decapeptide designated P3 (95-VGIGFHLQIY-104) that aligns with receptor binding sites of FGF-1 and FGF-2 suppressed FGF-5-induced proliferation of BALB/3T3 A31 and NIH/3T3 murine fibroblasts, and FGF receptor-1c (FGFR-1c)-transfected Ba/F3 cell line (FR-Ba/F3 cells). IC50s of this peptide on these cell proliferations were 64, 28, 146 microM, respectively. On the other hand, IC50 of this peptide on binding of FGF-5 to the FGFR-1(IIIc)/Fc chimera was 483 microM. Examination in dorsal depilated mice revealed that the P3 peptide reduced the activity of FGF-5 to recover hair pigmentation and hair follicle lengths. The classification of histologically observed skin sections showed FGF-5-induced delations of anagen procedure had reduced by the P3 peptide. The anti-Ki67 antibody staining of hair follicles was inhibited by administration of FGF-5, and this inhibition by FGF-5 was recovered by administration of the P3 peptide. The P3 peptide alone did not affect hair follicle length and hair cell proliferation. These results indicate that the decapeptide antagonized FGF-5 activity in vivo, and reduced the inhibition of FGF-5 in hair growth, confirming that FGF-5 inhibitors are promising substances against hair loss and/or for promoting hair growth.     

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.     

Basic fibroblast growth factor (FGF-2) protects rat cochlear hair cells in organotypical culture from aminoglycoside injury

Given the evidence that basic fibroblast growth factor (FGF-2) can protect neural and retinal cells from degeneration, we evaluated the potential of this growth factor to protect sensory cells in the inner ear. When sensory cells of the organ of Corti are exposed to aminoglycoside antibiotics such as neomycin either in vivo or in vitro, significant ototoxicity is observed. The in vitro cytotoxic effects of neomycin are dose and time dependent. In neonatal rat organ of Corti cultures, complete inner and outer hair cell destruction is observed at high (mM) concentrations of neomycin while inner hair cell survival and severely damaged outer hair cells are noted at moderate (μM) concentrations, with a maximal effect observed after 2 days of culture. Approximately 50% of cochlear outer hair cells are lost at a dose of 35 μM neomycin, and most surviving cells show disorganized stereocilia. Inner hair cells show primarily disorganization of their stereocilia. A significant protective effect is observed when the organ of Corti is pre-treated with FGF-2 (500 ng/ml) for 48 hours, and then FGF-2 is included with neomycin in the culture medium. A greater extent of outer hair cell survival and a significant decrease in stereociliary damage are noted with FGF-2. However, disorganization of inner hair cell stereocilia is unaffected by FGF-2. The protective effect of FGF-2 is specific, since interleukin-1B, nerve growth factor, tumor necrosis factor, and epidermal growth factor are ineffective, while retinoic acid and transforming growth factor alpha show only a moderate protective effect. These results confirm the potential of molecules like FGF-2 for preventing cell death due to a variety of causes. © 1996 Wiley-Liss, Inc.     

Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.