Ken-ichi Matsuda

Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing.

Circulation‐derived cells play a crucial role in the healing processes of tissue. In early phases of tendon healing processes, circulation‐derived cells temporarily exist in the wounded area to initiate the healing process and decrease in number with time. We assumed that a delay of time‐dependent decrease in circulation‐derived cells could improve the healing of tendons. In this study, we injected platelet‐rich plasma (PRP) containing various kinds of growth factors into the wounded area of the patellar tendon, and compared the effects on activation of circulation‐derived cells and enhancement of tendon healing with a control group (no PRP injection). To follow the circulation‐derived cells, we used a green fluorescent protein (GFP) chimeric rat expressing GFP in the circulating cells and bone marrow cells. In the PRP group, the numbers of GFP‐positive cells and heat‐shock protein (HSP47; collagen‐specific molecular chaperone)‐positive cells were significantly higher than in the control group at 3 and 7 days after injury. At the same time, the immunoreactivity for types I and III collagen was higher in the PRP group than in the control group at early phase of tendon healing. These findings suggest that locally injected PRP is useful as an activator of circulation‐derived cells for enhancement of the initial tendon healing process. J. Cell. Physiol. 215: 837–845, 2008.

Colocalization of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus and hypothalamus.

We investigated the distribution and colocalization pattern of the two corticosteroid receptors, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the hippocampus and hypothalamus, the main target regions of corticosterone in the rat brain, using double immunofluorescence histochemistry in conjunction with specific polyclonal antibodies against MR and GR. In the hippocampus, MR- and GR-immunoreactivity (ir) were colocalized in CA1 and CA2 pyramidal neurons and granule cells of the dentate gyrus, while only MR-ir was seen in the CA3 pyramidal neurons. Colocalization of MR- and GR-ir was also observed in the parvocellular region, but not in the magnocellular region of the paraventricular nucleus (PVN). Subcellular distribution of MR-ir was more cytoplasmic in comparison with that of GR-ir, while the ratio of cytoplasmic to nuclear distribution of these receptors was different among the regions. After adrenalectomy (ADX), the distribution pattern of both receptors was changed to cytoplasmic, although the degree of the change of distribution was different among each region. Replacement of corticosterone after ADX recovered the distribution pattern to that of the sham-operated animals. These results suggest that the balance of MR and GR in the cell underlies the potential regulation of corticosteroid through the hippocampus and hypothalamus.

Expression and intracellular distribution of the G protein-coupled receptor 30 in rat hippocampal formation

Although the expression and distribution of nuclear estrogen receptors in the hippocampus has been described, it has been proposed that the nuclear receptors may not explain all aspects of estrogen function in the hippocampus. Recently, a G protein-coupled receptor for estrogen, GPR30, was identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we examined the expression and intracellular distribution of GPR30 in the rat hippocampal formation. We found expression of GPR30 in pyramidal cells of CA1-3 and granule cells of the dentate gyrus at both mRNA and protein levels. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was mainly localized to the Golgi apparatus and partially in the endoplasmic reticulum of the neuron but could not detect the protein at the cell surface. Expression levels were not different among male, female in proestrus and female in estrus at the adult stage, but were higher in newborn male than newborn female.

Behavior of Transplanted Bone Marrow-derived GFP Mesenchymal Cells in Osteochondral Defect as a Simulation of Autologous Transplantation

To elucidate the behavior of autologously transplanted mesenchymal cells in osteochondral defects, we followed transplanted cells using green fluorescent protein (GFP) transgenic rats, in which all cells express GFP signals in their cytoplasm and nuclei as transplantation donors. Bone marrow-derived mesenchymal cells, which contain mesenchymal stem cells (MSCs), were obtained from transgenic rats. Then, dense mesenchymal cell masses created by hanging-drop culture were transplanted and fixed with fibrin glue into osteochondral defects of wild-type rats. At 24 weeks after surgery, the defects were repaired with hyaline-like cartilage and subchondral bone. GFP positive cells, indicating transplanted mesenchymal-derived cells, were observed in the regenerated tissues for 24 weeks although GFP positive cells decreased in number with time. Because GFP causes no immunological rejection and requires no chemicals for visualization, transplantation between transgenic and wild-type rats can be regarded as a simulation of autologous transplantation, and the survivability of transplanted cells are able to be followed easily and reliably. Thus, the behavior of transplanted mesenchymal cells was able to be elucidated in vivo by this strategy, and the results could be essential in future tissue engineering for the regeneration of osteochondral defects with original hyaline cartilage and subchondral bone.

Sexually dimorphic gastrin releasing peptide system in the spinal cord controls male reproductive functions.

Neurons in the upper lumbar spinal cord project axons containing gastrin-releasing peptide (GRP) to innervate lower lumbar regions controlling erection and ejaculation. This system is vestigial in female rats and in males with genetic dysfunction of androgen receptors, but in male rats, pharmacological stimulation of spinal GRP receptors restores penile reflexes and ejaculation after castration. GRP offers new avenues for understanding potential therapeutic approaches to masculine reproductive dysfunction.

GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP‐positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation‐derived cells but not tendon‐derived cells. Tendon‐derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation‐derived cells had decreased in number and had been replaced by the tendon‐derived cells. These findings suggest that circulation‐derived and tendon‐derived cells contribute to the healing of tendons in different periods as part of a biphasic process. J. Cell. Physiol. 210: 684–691, 2007.

Visualization of Glucocorticoid Receptor and Mineralocorticoid Receptor Interactions in Living Cells with GFP-Based Fluorescence Resonance Energy Transfer

Adrenal corticosteroids readily enter the brain and exert markedly diverse effects, including stress responses in the target neural cells via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). It has been shown that the GR and MR are highly colocalized in the hippocampus. Given the differential action of the MR and GR in the hippocampal region, it is important to elucidate how these receptors interact with each other in response to corticosteroids. We investigated the heterodimerization of the MR and GR with green fluorescent protein-based fluorescence resonance energy transfer (FRET) microscopy in living cells with spatiotemporal manner. FRET was evaluated in three ways: (1) ratio imaging; (2) emission spectra; and (3) acceptor photobleaching. FRET analysis demonstrated that cyan fluorescent protein-GR and yellow fluorescent protein-MR form heterodimers after corticosterone (CORT) treatment both in the nucleus of cultured hippocampal neurons and COS-1 cells, whereas they do not form heterodimers in the cytoplasm. The content of the GR-MR heterodimer was higher at 10-6 m CORT than at 10-9 m CORT and reached a maximum level after 60 min of CORT treatment in both cultured hippocampal neurons and COS-1 cells. The distribution pattern of heterodimers in the nucleus of cultured hippocampal neurons was more restricted than that in COS-1 cells. The present study using mutant fusion proteins in nuclear localization signal showed that these corticosteroid receptors are not translocated into the nucleus in the form of heterodimers even after treatment with ligand and thus allow no heterodimerization to take place in the cytoplasm. These results obtained with FRET analyses give new insights into the sites, time course, and effects of ligand concentration on heterodimersization of the GR and MR.

Bone marrow-derived cells from the footprint infiltrate into the repaired rotator cuff

BACKGROUND Cells from the bone marrow are considered important during the rotator cuff repair process, but the kinetics of bone marrow-derived cells in this process is unknown. PURPOSE To analyze the kinetics of bone marrow cells during the rotator cuff repair process, to review whether or not they are histologically involved in rotator cuff healing, and to analyze the biomechanics of the repaired tissues. METHODS Bone marrow chimeric rats that express green fluorescent protein (GFP) only in bone marrow- and circulation-derived cells were created. Bilateral supraspinatus tendons were separated from the greater tuberosity of the humeral head to produce a rotator cuff transection model. Drilling into the bone marrow was performed in the greater tuberosity of the right humerus and the supraspinatus tendon was repaired (drilling group), while the supraspinatus tendon was repaired on the left shoulder without drilling (control group). We examined the histology of the rotator cuff, the ultimate force-to-failure, and the proportion of GFP-positive cells in the repaired rotator cuff at 2, 4 and 8 weeks after surgery. RESULTS Mesenchymal cells were observed in the repaired rotator cuff at 2 weeks in both groups. There were more GFP-positive cells in the drilling group than the control group at 2, 4 and 8 weeks. The ultimate force-to-failure was significantly higher in the drilling group than the control group at 4 and 8 weeks. CONCLUSION Bone marrow-derived cells passed through holes drilled in the humerus footprint, infiltrated the repaired rotator cuff and contributed to postsurgical rotator cuff healing.

Keratinocyte Growth Inhibition through the Modification of Wnt Signaling by Androgen in Balding Dermal Papilla Cells

CONTEXT/OBJECTIVE Androgen induces androgenetic alopecia (AGA), which has a regressive effect on hair growth from the frontal region of the scalp. Conversely, Wnt proteins are known to positively affect mammalian hair growth. We hypothesized that androgen reduces hair growth via an interaction with the Wnt signaling system. The objective of this study was to investigate the effect of androgen on Wnt signaling in dermal papilla (DP) cells. DESIGN The effect of androgen and Wnt3a on keratinocyte proliferation was measured by use of a coculture system consisting of DP cells and keratinocytes. The molecular mechanisms of androgen and Wnt pathway interactions in DP cells were examined by analyzing the expression, intracellular localization, and activity of the androgen receptor (AR) and also downstream Wnt signaling molecules. RESULTS Wnt3a-dependent keratinocyte growth was suppressed by the addition of dihydrotestosterone in coculture with DP cells that were derived from AGA patients, but growth was not suppressed in coculture with DP cells from non-AGA males. Whereas DP cells from both scalp regions expressed AR protein, the expression levels of AR and cotranslocation with beta-catenin, a downstream Wnt signaling molecule, were higher in DP cells of AGA patients than in DP cells from non-AGA males. In addition, significant suppression of Wnt signal-mediated transcription in response to dihydrotestosterone treatment was observed only in DP cells from AGA patients. CONCLUSION These results suggest that Wnt signaling in DP cells is regulated by androgen and this regulation plays a pivotal role in androgens action on hair growth.

Characterization of Nuclear Import of the Domain-Specific Androgen Receptor in Association with the Importin α/β and Ran-Guanosine 5′-Triphosphate Systems

Androgen induces androgen receptor (AR) nuclear import, which allows AR to act as a transcriptional factor and ultimately leads to biological activity. However, the mechanism of AR translocation to the nucleus is still unclear. In the present study, we assessed the nuclear import abilities of each domain of AR and their mechanisms related to Ran and importin alpha/beta using green fluorescent protein real-time imaging. The localization of AR to the nucleus in the absence and presence of ligands was dependent upon a complex interplay of the amino terminal transactivation domain (NTD), the DNA binding domain (DBD), and the ligand binding domain (LBD). NTD and DBD showed ligand-independent nuclear import ability, whereas LBD had ligand-dependent transport. In addition, AR deletion mutant lacking DBD was distributed in the cytoplasm regardless of ligand existence, suggesting that the remaining domains, NTD and LBD, are responsible for AR cytoplasmic localization. Cotransfection with a dominant negative form of Ran dramatically inhibited the nuclear import of all AR domains, and a dominant negative form of importin alpha prevented AR and DBD import. Importin beta-knockdown strongly blocked DBD import. These results indicate that there are two additional nuclear localization signals (NLSs) in the NTD and LBD, and there are distinct pathways used to attain domain-specific AR nuclear import: the NLS of DBD is Ran and importin alpha/beta-dependent, whereas the NLSs of NTD and LBD are Ran dependent but importin alpha/beta-independent. Our data suggest that the nuclear import of AR is regulated by the interplay between each domain of the AR.