Epithelial appendage development requires complex cell signaling and interaction both between epithelial cells and between epithelia and mesoderm-derived dermis (Sengel, 1983;Chuong et al, 1996,2001). Secretory signals are believed to be a key component in communication and regulation of cell proliferation, growth, differentiation, and formation of epithelial appendages (McElwee and Hoffmann, 2000).
Hepatocyte growth factor (HGF) has previously been identified as a powerful modulator of hair growth and may play an important role in hair follicle development and cycling (Lindner et al, 2000). Macrophage-stimulating protein (MSP) is another member of the HGF family of growth factors and is also known as hepatocyte growth factor-like protein (HGFL) and scatter factor 2. MSP was initially identified as a factor that promotes chemotactic responses in peritoneal resident macrophages (Leonard and Skeel, 1976,1979;Leonard, 1997). More recently, MSP has been shown to induce keratinocyte growth (Wang et al, 1996a), liver cell morphogenesis, and motogenesis (Medico et al, 1996), prevent apoptosis of epithelial cells separated from the extracellular matrix (Danilkovitch et al, 2000;Danilkovitch-Miagkova and Leonard, 2001), promote bone resorption by osteoclast cells (Kurihara et al, 1996,1998), and act as a neurotrophic factor (Funakoshi and Nakamura, 2001;Stella et al, 2001).
MSP communicates with cells through binding a cell surface receptor tyrosine kinase called RON (Receptuer d'Origine Nantaise) in humans (also called macrophage stimulating 1 receptor; MST1R) or STK in mice as distinct from the cell receptor for HGF named MET (Ronsin et al, 1993;Wang et al, 1994b,1995). RON is expressed during embryogenesis primarily in the central and peripheral nervous systems, developing bones, skin, lung, and digestive tract epithelia (Gaudino et al, 1995;Quantin et al, 1995;Thierry et al, 1995). This expression during development suggests the RON receptor and its ligand MSP are involved in the proliferation and development of epithelial tissues, bones, and neuroendocrine derivatives (Gaudino et al, 1995). RON is also known to be expressed on mature peritoneal macrophages, endothelium, and epithelia and may be upregulated in response to tissue injury (Iwama et al, 1995;Sakamoto et al, 1997;Nanney et al, 1998;Cowin et al, 2001). This relatively restricted expression of RON/STK is in contrast to the HGF receptor MET that is broadly expressed and mediates pleiotropic biological functions such as cell growth, motility, and morphogenesis (Sonnenberg et al, 1993).
Because of MSP's close relationship to HGF, the distribution of its receptor RON in human hair follicles was examined and the effects of MSP on hair growth were evaluated with functional studies. The results demonstrated a significant hair growth-promoting impact for MSP on hair follicles.
Results
MSP promotes growth of human hair follicles ex vivo
Culture of human hair follicles with MSP demonstrated a net increase in hair growth rates in those follicles exposed to MSP as compared with controls. Little difference in the hair follicle growth rates was observed between the five donors and the results were combined (Figure 1). Using the micrometer measurements from day 8 of culture, the statistical significance of increased hair growth only reached the 95% confidence interval in follicles exposed to 1 ng of MSP as compared with controls. Probability values of 0.255, 0.036, 0.156, and 0.263 were obtained for hair follicles exposed to 0.1, 1, 10, and 100 ng per mL MSP, respectively.
Figure 1.
Human hair follicle growth was accelerated in response to macrophage-stimulating protein (MSP) exposure. Isolated hair follicles incubated in the presence of 0.1, 1, 10, or 100 ng per mL of MSP over 8 d all demonstrated a degree of increased growth as compared with controls.
Full figure and legend (26K)MSP induces anagen in telogen stage hair follicles in vivo
Previous studies have demonstrated recombinant human MSP can successfully stimulate mouse keratinocytes (Wang et al, 1996a). Here, recombinant human MSP was examined for its ability to modulate hair growth in C3H/HeJ mice. All eight 70-d-old mice implanted with agarose beads containing 1 
g of MSP exhibited a marked response with hair growth visible in skin immediately above the location of the beads (Figure 2a). Hair growth was first observed on day 16 post-implantation simultaneously in all mice exposed to MSP. The second group of eight mice receiving 100 ng of MSP in the same volume of agarose beads showed a more limited response with only four of eight demonstrating overt hair growth at the site of bead implantation by necropsy (not shown). In contrast, eight control mice exhibited no hair growth over the bead implantation site or elsewhere in the shaved dorsal region on day 16 and this state was maintained until necropsy, 24 d after bead implantation (Figure 2b). Of eight mice that were not implanted with beads and monitored for the onset of spontaneous anagen, four showed a change in skin color, an indication of anagen onset, on part of the shaved dorsal surface when aged 104 d. All eight mice had dorsal skin containing anagen stage follicles when aged 110 d. Histology at necropsy demonstrated hair follicles located over beads incubated with 1 g MSP were in anagen whereas adjacent skin contained telogen stage hair follicles (Figure 3a). Histology also confirmed that control mice presented a uniform telogen hair follicle state (Figure 3b).
Figure 2.
Implantation of agarose beads incubated with macrophage-stimulating protein (MSP)-induced anagen in telogen stage pelage follicles. Agarose beads incubated with MSP were injected subcutaneously to female mice aged 70 d when dorsal pelage follicles are in a uniform telogen state. By day 92 mice receiving 1 g of MSP all exhibited some degree of anagen hair growth over the bead implantation site (A) whereas control mice demonstrated no hair growth over or beyond the site of bead implantation (B).
Figure 3.
Exposure to macrophage-stimulating protein (MSP)-induced anagen in telogen stage hair follicles and enabled prolongation of anagen in a small subset of hair follicles. At necropsy, mice aged 94 d implanted with MSP-soaked beads demonstrated anagen stage hair follicles in the immediate vicinity of the beads (A) whereas control mice exhibited uniform telogen stage follicles (B). Mice age 20 d exposed to MSP presented with a few isolated hair follicles in an anagen state, most pronounced when associated with beads that had penetrated to an intradermal location (C). In contrast, control mice had a uniform telogen state over subcutaneous and intradermally located beads (D). Scale bar=500 M.
All five aged C3H/HeJ mice receiving 1 g of MSP demonstrated a hair growth response over the site of bead implantation and also exhibited hair growth beyond the location of the beads. Hair growth was apparent simultaneously in all mice by day 13 post-bead implantation, although control mice did not demonstrate visible hair growth at this stage. Of five control mice, four revealed limited hair growth in the shaved dorsal region, but not over or immediately adjacent to the bead implantation site, whereas one mouse had anagen hair growth over and beyond the bead implantation site by day 17. These states persisted until necropsy and all mice employed survived until necropsy. Histology of the aged mice revealed that hair follicles in skin above beads soaked in MSP were still in an anagen state at necropsy whereas hair follicles beyond the bead location were in telogen. Histology of control mice revealed a telogen state in hair follicles above and immediately adjacent to the site of bead implantation including the mouse with apparent spontaneous hair growth over the bead implantation site.
Mice implanted with agarose beads containing 1 g of recombinant human MSP at 10 d of age and necropsied when 20 d old revealed that a minority of pelage hair follicles above the location of the beads were maintained in an anagen state whereas hair follicles in pelage skin adjacent to the location of the beads were uniformly in telogen (Figure 3c,d). Comparative control mice exhibited a uniform telogen in all pelage hair follicles over the site of bead location and adjacent to it.
All mice employed in the studies described demonstrated no apparent abnormalities or responses to agarose bead implantation other than those described above. Although MSP is known as a stimulator of peritoneal macrophage cells, no apparent inflammation at the site of MSP-soaked agarose bead implantation was observed. Control mice receiving agarose beads not exposed to MSP were also devoid of inflammation. Other than the presence of agarose beads in a subcutaneous location and a hair growth response to MSP exposure, no other morphological abnormalities were apparent in the skin of study mice.
RON receptor is expressed in the hair follicle epithelium and mesenchyme and mature MSP is present in the hair follicle mesenchymal component
Using an antibody reactive against the human MSP receptor RON, we identified expression of the receptor predominantly located in the inner root sheath of anagen stage human hair follicles as well as the outer root sheath, differentiating hair matrix, DP and some cells of the DSC (Figure 4). A low level of expression in keratinocytes of the non-follicular epithelium was also observed. Apparent expression of the RON receptor in the arrector pili muscle was shown to be largely non-specific by peptide depletion controls. The anti-MSP antibody described failed to identify tissue MSP expression suggesting the unsuitability of this antibody for immunohistological techniques. Consequently, a western blotting approach was employed and indicated consistent presence of mature, bioactive MSP in both the cytosol and membrane fractions from cultured DP-derived cells and to a lesser extent in DSC-derived cells. The mature form of MSP was not observed in DS-derived cells, only the pro-MSP precursor was identified in the DS cell membrane fraction. (Figure 5).
Figure 4.
Macrophage-stimulating protein (MSP) receptor Receptuer d'Origine Nantaise (RON) is broadly expressed in human hair follicles. Sections of normal human scalp skin were processed for peptide–antibody depleted controls (A, C) in comparison with RON receptor distribution by immunohistology (B, D–H). As compared with peptide control (A), presence of RON in hair follicle outer root sheath was most striking (B). Peptide–antibody depleted control tissue samples (C) revealed that apparent intense expression in the arrector pili muscle in test tissues (D) was likely non-specific binding of the antibody. Slight, but specific RON expression was observed in the basal layer of non-follicular epithelium (E). RON expression was present with greater intensity in the outer and inner root sheath and hair matrix (F, G) although expression was also apparent to a lesser extent in the dermal papilla and some dermal sheath cup cells, of anagen stage hair follicles (H). Scale bars=100 M.
Figure 5.
Macrophage-stimulating protein (MSP) is present in cultured dermal papilla (DP) and dermal sheath cup (DSC) cells. Western blotting using recombinant human MSP as a positive control (lane 1) and extracts from cultured DP (cytosol, lane 2; membrane, lane 5), DSC (cytosol, lane 3; membrane, lane 6), and DS (cytosol, lane 4; membrane, lane 7), derived cells. Pro-MSP, the single-chain inactive precursor of mature active MSP, was observed in membrane fractions from all three cell types, DP (lane 5), DSC (lane 6), and DS (lane 7) as an 80 kDa band. The membrane fractions of DP and DSC, but not DS cells also exhibited a band at 56 kDa for the 
chain of mature MSP and with greatest intensity in DP cell membranes. Cytosol extracts from DP (lane 2) and to a lesser extent DSC (lane 3) cells exhibited presence of an MSP chain band. In contrast, the cytosol of DS cells did not contain the mature form of MSP (Lane 4).
Discussion
As a member of the HGF family of cytokines, and given HGF is known to modulate hair follicle growth among many other properties (Jindo et al, 1998;Lindner et al, 2000), MSP was investigated for its respective action on hair follicles. MSP acts on cells through binding a cell surface receptor tyrosine kinase called RON in humans or STK in mice as distinct from the cell receptor for HGF named MET (Ronsin et al, 1993;Wang et al, 1994b,1995). Functional absence of MSP in knockout mice has little immediately noticeable impact (Bezerra et al, 1998), although the parameters of hair growth in such mice is not known. In contrast, complete absence of RON/STK expression in knockout mice leads to early embryonic death demonstrating a fundamental requirement for RON and suggesting that in addition to MSP there are other, as yet unknown, factors that can ligand with RON/STK (Muraoka et al, 1999).
RON/STK receptors act via a two-phosphotyrosine docking site, capable of concomitant activation of multiple intracellular transducers and signalling pathways (Tamagnone and Comoglio, 1997). RON/STK mRNA is translated into a glycosylated precursor that is cleaved into a 185-kDa heterodimer of 35-kDa () and 150-kDa (
) subunits joined by disulfide linkage. HGF stimulation has no effect on RON receptor activation, but upon stimulation by MSP the chain of RON/STK undergoes tyrosine phosphorylation (Gaudino et al, 1994). Both the MSP and chain heterodimer and the chain alone can bind to the RON receptor, but only the heterodimeric MSP induces RON/STK receptor dimerization and activation (Leonard, 1997;Tamagnone and Comoglio, 1997;Wang et al, 1997). Downstream events after receptor liganding are poorly understood, but studies suggest the RON receptor may regulate phosphorylation of catenin (Danilkovitch-Miagkova et al, 2001), a key factor in hair follicle morphogenesis and growth (Gat et al, 1998). Immunohistology defined expression of the RON/STK receptor for MSP to be dominant within keratinocyte cells of the anagen stage hair follicle in humans. Other studies suggest that some degree of RON/STK may be expressed on most keratinocyte cells (Wang et al, 1996a) and we observed low levels of positive staining by immunohistology in the non-follicular epidermis. However, more intense expression of RON/STK in epidermal appendages indicates these structures are most likely to respond to MSP and MSP may elicit the greatest response in cells with high RON/STK expression.
MSP is an 80 kDa disulfide-linked heterodimeric protein containing a 452 amino acid residue chain and a 228 amino acid chain with molecular masses of 52 and 25 kDa, respectively (Yoshikawa et al, 1999). Due to glycosylation, the and chains migrate as 56 and 30 kDa proteins in SDS-PAGE (Skeel et al, 1991;Yoshimura et al, 1993,1999). Like HGF, MSP is secreted as a single-chain inactive precursor pro-MSP. It is converted to an active heterodimer form through proteolytic cleavage by kallikrein, factor XIIa, factor XIa, nerve growth factor-
(NGF-), and epidermal growth factor-binding protein (EGF-BP), the serine protease subunits of NGF and EGF (Wang et al, 1994a–c). This suggests possible cooperative interaction between NGF- or EGF-BP and pro-MSP in inflammation and wound healing (Wang et al, 1994a;Nanney et al, 1998). Western blotting against cultured cells from the DP, DSC, and DS mesenchymal hair follicle components demonstrated highest expression of mature MSP in cell membrane and cytosol fractions of DP cells and lowest expression in DS cells, while expression in DSC cells was intermediate. This presence of mature MSP suggests the potential of the hair follicle mesenchymal component to cleave pro-MSP into a mature, active form, and possibly to signal to the epithelium-derived component.
The ex vivo results demonstrated that MSP can stimulate increased rates of hair growth in excess of that seen in hair follicles not exposed to MSP in the first 8 d of hair follicle culture. A superior accelerated growth rate was obtained with an MSP concentration in the range of 1 ng per mL, a concentration similar to that defined for ex vivo studies using HGF (Jindo et al, 1994,1995;Shimaoka et al, 1995). Implantation to C3H/HeJ mice of agarose beads soaked in human recombinant MSP demonstrated that MSP has a limited capability to prolong the anagen growth phase of the primary pelage coat generation in young mice, complementing the results of ex vivo human hair follicle culture studies. Implantation of MSP incubated agarose beads to mice with dorsal telogen stage hair follicles were also capable of inducing an anagen growth state until necropsy. As such, MSP may be involved in modulating hair growth and analogs may have potential for treating alopecias in which there is an increase in the frequency of telogen stage hair follicles and low levels of hair growth.
Materials and Methods
MSP action on human hair follicles ex vivo
Protocols, similar to those used in previously published research on HGF (Jindo et al, 1995;Shimaoka et al, 1995), were employed to define the functional effects of MSP on human hair follicles. Five healthy human volunteers (two females, three males, mean age 27 y) at the Philipp University Department of Dermatology provided occipital scalp skin samples, with informed consent and according to Hospital Ethics Committee regulations, from which hair follicles were isolated by removing the epidermis and "plucking" the hair follicles with forceps from the dermis (Eicheler et al, 1998). Whole hair follicles were placed in Williams E medium, supplemented with insulin (10 g per mL), L-glutamine (2 mM), penicillin (100 U per mL), and streptomycin (100 g per mL) (Gibco Invitrogen GmbH, Karlsruhe, Germany) in 24-well culture plates (Falcon, Franklin Lakes, New Jersey) incubated at 37°C in 5% CO2. Hair follicles from each donor were separated into groups and 0.1, 1, 10, and 100 ng per mL recombinant human MSP (R&D systems, Minneapolis, Minnesota) in phosphate-buffered saline (PBS) was added whereas control follicles received PBS vehicle alone. Hair follicle length was measured on day 0 using a Nikon inverted microscope with a digital camera and LuciaM software (version 2.995
, Nikon GmbH, Düsseldorf, Germany) and at regular intervals after initiation of culture. Medium including MSP was replaced twice during the 8-d analysis period. Follicles that exhibited catagen, with dissecting microscope examination, or presented with no growth or an unusually high growth rate, indicative of a follicle in an early anagen state, were excluded from final analysis. Thirty-six control follicles were compared with 16 exposed to 0.1 ng, 30 receiving 1 ng, 18 with 10 ng, and 18 exposed to 100 ng of MSP. Hair growth on day 8 of in vitro culture with each MSP concentration was compared with the control group using two-tailed t tests.
In vivo analysis of MSP
To characterize the effects of MSP on hair follicles in vivo, recombinant human MSP was administered to mice in various states of hair growth. Normal haired C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, Maine) were utilized in all studies. Mice received conventional low soy oil diet (Altromin 1434, Altromin GmBH, Lage, Germany) and acidified water (pH 2.8–3.0) ad libitum for the duration of the study.
Agarose bead preparation
Agarose beads were prepared based on previously published protocols (Botchkarev et al, 2001). Briefly, 250 L of agarose beads in suspension as supplied (100–200 mesh (80–150 M), Bio Rad Laboratories Inc., Hercules, California) were extensively washed with sterile PBS (Gibco Invitrogen) and recombinant human MSP (R&D systems) at a concentration of 5 g per mL in PBS plus 0.5% C3H/HeJ mouse serum was added in a quantity of 1 g or 100 ng per 250 L of original agarose bead suspension. The agarose beads were incubated for 1 h at 37°C. For a comparative control, agarose beads were prepared in the same way, but were incubated with PBS plus 0.5% C3H/HeJ mouse serum alone. The agarose beads and solution were drawn into a 1 mL syringe with a 22-gauge needle and additional sterile PBS was added to permit injection of 150 L of the agarose bead suspension per mouse.
Agarose bead implantation
To characterize the ability of MSP to prolong the anagen growth phase of pelage hair follicles, eight mice aged 10 d old, when pelage hair follicles are in anagen of the first pelage coat generation (Dry, 1926), were implanted with a single injection of agarose beads containing 1 g of recombinant human MSP per injection. Eight littermates received agarose beads in suspension in the absence of MSP. The mice were observed for a further 10 d and necropsied when 20 d old when generation of the first pelage coat is normally complete and pelage hair follicles of the dorsal skin are typically in telogen (Dry, 1926).
To characterize the ability of MSP to induce anagen in telogen stage hair follicles, eight mice all aged 70 d, when the dorsal pelage hair follicles are in a telogen resting state after completion of the second coat generation (Dry, 1926), were each injected with agarose beads in suspension providing 1 g of MSP per mouse. A further eight mice were injected with the same volume of agarose beads providing 100 ng of MSP per mouse. Eight age-matched mice received agarose beads unexposed to MSP as a control. The 24 mice were observed for 24 d and all were necropsied aged 94 d. A further eight mice of the same age did not receive agarose beads and were observed indefinitely to define the spontaneous onset of anagen.
To further characterize the effects of MSP on hair follicles in aging C3H/HeJ mice, a large number of mice all aged 246 d were shaved on their dorsal surface and those mice exhibiting a uniform telogen stage skin were selected for use. Five mice were injected with agarose beads in suspension providing 1 g of MSP per mouse. Five mice served as controls receiving beads not exposed to MSP. The mice were observed for 24 d and all were necropsied aged 270 d.
Dorsal skin samples, including the site of bead implantation and skin surrounding the implantation, were fixed in Fekete's acid–alcohol–formalin solution, paraffin embedded, sectioned at 6 M through the area of bead implantation, and stained with hematoxylin and eosin for histologic evaluation (Relyea et al, 1999).
Immunohistology
Immunohistology was performed on 6 M cryostat tissue sections of human hair follicles. Briefly, tissue alkaline phosphatase acivity was ablated with levamisole solution and non-specific binding blocked using an avidin–biotin blocking kit (Vector Laboratories Inc., Burlingame, California) and 2% normal goat serum. Immunohistology for the MSP receptor RON was conducted using a polyclonal antibody against human RON (RON (N-20), sc-14627, Santa Cruz Biotechnology Inc., Santa Cruz, California). Biotinylated swine anti-goat (Cedarlane Labs., Hornby, Ontario), alkaline phostphatase-conjugated avidin–biotin complex (Vector Laboratories Inc.), and fast red development substrate (Pierce Chemical Co., Rockford, Illinois) solutions were applied in sequence. Tissue sections were counterstained with Mayer's Hematoxylin. The primary antibodies were replaced with species and isotype-matched serum for negative controls. In addition, the peptide sequence (Santa Cruz) to which the RON-specific antibody was raised, was used in peptide depletion controls according to manufacturer's instructions. After preliminary screens to identify appropriate antibody dilution parameters, the procedure repeated on more than ten separate occasions for each antibody using multiple tissue sections from three different human donors (two males, one female).
Western blotting MSP expression by the hair follicle mesenchymal component
Human hair follicles from two donors were dissected and the dermal papilla (DP), peri-bulbar dermal sheath "cup" (DSC), and non-bulbar dermal sheath (DS) mesenchymal components were isolated for culture as described elsewhere (Eicheler et al, 1998). All cells regardless of source were cultured under the same conditions. Hair follicle subunits derived from an individual donor were maintained as separate cultures. DP, DS, or DSC units were placed in 1 mL of AmnioMax–C100 basal medium plus AmnioMax–C100 supplement (Gibco Invitrogen) in 24-well culture plates (Falcon) incubated at 37°C in 5% CO2. Culture conditions and medium were such that any contaminating keratinocytes were non-proliferative as confirmed by in vitro observation. Proliferating DP-, DS-, or DSC-derived cells were subsequently passaged a maximum of two times into 25 mL culture flasks (Greiner, Frickenhausen, Germany) to produce 1 
107+ cells per culture.
The cell cytosol and membranes were fractionated by physical dissociation of cells in lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1 mM ethylenediamine tetraacetic acid, 10 mM MgCl2, 3 mM dithiothreitol, 50 mM NaCl, 17.2 mM phenylmethylsulfonyl fluoride) and centrifugation at 300,000 g for 30 min at 4°C. Membrane and cytosol fractions from DP, DSC, and DS cells were subsequently resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE: 12% polyacrylamide) using 20 g of extract per lane. For western blots, gels were transferred to membranes (Hybond ECL, Amersham Biosciences GmbH, Freiburg, Germany) using a semi-dry blotting apparatus. Membranes were blocked with 5% non-fat milk powder in PBS overnight at 4°C and washed with 0.5% Tween 20 (Sigma-Aldrich GmbH, Munich, Germany) in PBS. Polyclonal goat anti-human antibody specific for the MSP chain was utilized to detect both the pro-MSP precursor and mature form of MSP (HGFL (N-19), sc-6088, Santa Cruz). Recombinant human MSP (R&D systems) was employed for a positive control and peptide depletion (Santa Cruz) was utilized as a negative control. Anti goat HRP-conjugated secondary antibody (Amersham) and ECL detection reagents (Amersham) prepared as per manufacturers instructions, were applied in sequence.
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Acknowledgments
K. J. M. is a recipient of the Alfred Blaschko memorial fellowship, Marburg. R. H. is a recipient of the William J. Cunliffe award.
