A Vitronectin-Derived Peptide Restores Ovariectomy-Induced Bone Loss by Dual Regulation of Bone Remodeling.

BACKGROUND: Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. METHODS: We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6 mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4 weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. RESULTS: VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3 weeks. CONCLUSION: Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.

A vitronectin-derived peptide prevents and restores alveolar bone loss by modulating bone re-modelling and expression of RANKL and IL-17A.

AIM: This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. MATERIALS AND METHODS: We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n = 10/group). VnP-16 (100 µg/10 µl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 µg/5 µl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. RESULTS: VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. CONCLUSIONS: Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

The laminin-211-derived PPFEGCIWN motif accelerates wound reepithelialization and increases phospho-FAK-Tyr397 and Rac1-GTP levels in a rat excisional wound splinting model.

We previously reported that the PPFEGCIWN motif (Ln2-LG3-P2-DN3), residues 2678-2686 of the human laminin α2 chain, promotes cell attachment of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs); however, its in vivo effects on cutaneous wound healing have not yet been examined. In this study, we sought to determine whether Ln2-LG3-P2-DN3 could promote full-thickness cutaneous wound healing by accelerating wound reepithelialization and wound closure in vivo. Ln2-LG3-P2-DN3 had significantly higher cell attachment and spreading activities than vehicle or scrambled peptide control in both NHEKs and NHDFs in vitro. The wound area was significantly smaller in rats treated with Ln2-LG3-P2-DN3 than in those treated with vehicle or scrambled peptide in the early phase of wound healing. Furthermore, Ln2-LG3-P2-DN3 significantly accelerated wound reepithelialization relative to vehicle or scrambled peptide and promoted FAK-Tyr397 phosphorylation and Rac1 activation. Collectively, our findings suggest that the PPFEGCIWN motif has potential as a therapeutic agent for cutaneous regeneration via the acceleration of wound reepithelization and wound closure.

A laminin-211-derived bioactive peptide promotes the osseointegration of a sandblasted, large-grit, acid-etched titanium implant.

Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.

A Vitronectin-Derived Bioactive Peptide Improves Bone Healing Capacity of SLA Titanium Surfaces.

In this study, we evaluated early bone responses to a vitronectin-derived, minimal core bioactive peptide, RVYFFKGKQYWE motif (VnP-16), both in vitro and in vivo, when the peptide was treated on sandblasted, large-grit, acid-etched (SLA) titanium surfaces. Four surface types of titanium discs and of titanium screw-shaped implants were prepared: control, SLA, scrambled peptide-treated, and VnP-16-treated surfaces. Cellular responses, such as attachment, spreading, migration, and viability of human osteoblast-like HOS and MG63 cells were evaluated in vitro on the titanium discs. Using the rabbit tibia model with the split plot design, the implants were inserted into the tibiae of four New Zealand white rabbits. After two weeks of implant insertion, the rabbits were sacrificed, the undecalcified specimens were prepared for light microscopy, and the histomorphometric data were measured. Analysis of variance tests were used for the quantitative evaluations in this study. VnP-16 was non-cytotoxic and promoted attachment and spreading of the human osteoblast-like cells. The VnP-16-treated SLA implants showed no antigenic activities at the interfaces between the bones and the implants and indicated excellent bone-to-implant contact ratios, the means of which were significantly higher than those in the SP-treated implants. VnP-16 reinforces the osteogenic potential of the SLA titanium dental implant.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/- cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/- cells, but only at concentrations of 100 µg/ml and greater, and only in the presence of S9 activation. Pig-a mutations in L5178Y/Tk+/- cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G→A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

A laminin-derived functional peptide, PPFEGCIWN, promotes bone formation on sandblasted, large-grit, acid-etched titanium implant surfaces.

PURPOSE: This study aimed to investigate the in vitro and in vivo bone-forming potential of a sandblasted, large-grit, acid-etched (SLA) titanium (Ti) surface treated with a laminin-derived functional peptide, PPFEGCIWN (DN3). MATERIALS AND METHODS: Human osteoblast-like MG63 cells were cultured with SLA Ti discs untreated or treated with DN3 or a control scrambled peptide (SP). Cell adhesion, spreading, and viability on the discs were tested. Alkaline phosphatase gene expression and enzyme activity were also evaluated. Four DN3-coated SLA Ti implants and four untreated implants were placed into the tibiae of two rabbits (two implants/tibia). Ten days later, the bone-implant interfaces were subjected to histomorphometry to measure the bone response. The surface properties of the discs and implants were determined using scanning electron, widefield confocal, and confocal laser microscopy and x-ray photoelectron spectroscopy. RESULTS: The peptide-treated and untreated discs and implants were similar in terms of physical surface properties, but the peptide-treated surfaces had significantly higher nitrogen levels (P < .05). The DN3 peptide promoted cell adhesion, spreading, and alkaline phosphatase expression and enzyme activity (P < .05). Histomorphometry of the harvested implants showed rapid bone formation and affinity of the motif. CONCLUSION: This study suggests that treatment with the cell adhesion peptide DN3 promotes bone healing at the SLA Ti surface.

A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation.

Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with ß1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvß3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.

Functional diversity of miR-146a-5p and TRAF6 in normal and oral cancer cells.

Numerous studies implicate miR-146a as pleiotropic regulator of carcinogenesis; however, its roles in carcinogenesis are not fully understood. A clue from expression analyses of miR-146a-5p in all 13 oral squamous cell carcinoma (OSCC) cell lines examined and in OSCC tissues, whole blood and whole saliva of OSCC patients in vivo revealed that miR­146a-5p expression was highly upregulated. Particularly, we widened the view of its upregulation in saliva, implicating that high miR-146a-5p expression is not only correlated closely to the development of human oral cancer, but also to a possible candidate as a diagnostic marker of OSCC. Indeed, further examination showed that exogenous miR-146a-5p expression showed pleiotropic effects on cell proliferation and apoptosis which were partially based on the contextual responses of activation of JNK, downstream of TRAF6 that was targeted by miR-146a-5p in normal human keratinocytes and OSCC cell lines. TRAF6 suppression by a TRAF6-specific siRNA resulted in contradictory consequences on cellular processes in normal and OSCC cells. Notably, TRAF6 downregulation by both miR-146a-5p and TRAF6-specific siRNA deactivated JNK in SCC-9, but not in normal human keratinocytes. In support of the proliferation-promoting effect of miR-146a-5p, silencing of endogenous miR-146a-5p significantly reduced proliferation of SCC-9. Together, these results suggest that miR-146a-5p affects proliferation and apoptosis in a cellular context-dependent manner and selectively disarms the TRAF6-mediated branch of the TGF-ß signaling in OSCC cell lines by sparing Smad4 involvement.

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering.

Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.

A transcriptional roadmap to the senescence and differentiation of human oral keratinocytes.

Human epithelial cells undergo morphological and molecular changes leading to terminal differentiation and replicative senescence after a finite number of cell divisions during serial subculture. However, the target genes and their functional significance in the senescence and differentiation in normal human oral keratinocytes have been poorly defined. Here, we demonstrated normal human oral keratinocytes transcriptional signature profiling to senescence and differentiation. Using microarray analysis, our findings indicated that the gene expression profiles induced by serial subculture are distinct classes of gene. The greatest number of these altered genes was identified as being related to biological pathways of transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response, suggesting that the serial subculture is able to induce a multitude of specific gene expression changes during senescence and differentiation. Several highly upregulated genes (IL-1ß, S100A8, S100A9, MMP1, MMP9, IL-8, BHLHB2, HES1, and TWIST1) in response to the serial subculture in normal human oral keratinocytes were observed. In vitro and in vivo studies also exhibited a close relationship between senescence and differentiation of primary oral keratinocytes and expression of inflammatory molecules. These results suggest a new approach to determine the biological events underlying the pathogenesis of oral keratinocyte aging.

Titanium surface coating with a laminin-derived functional peptide promotes bone cell adhesion.

Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration.

Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.

Protein kinase Cα/ß inhibitor Gö6976 promotes PC12 cell adhesion and spreading through membrane recruitment and activation of protein kinase Cδ.

Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/ß. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 µM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/ß signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.

The potential of laminin-2-biomimetic short peptide to promote cell adhesion, spreading and migration by inducing membrane recruitment and phosphorylation of PKCδ.

Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3ß1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3ß1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/ß inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3ß1 integrin, and that binding of α3ß1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.

The potential of mouse skin-derived precursors to differentiate into mesenchymal and neural lineages and their application to osteogenic induction in vivo.

Although previous studies indicate that skin-derived precursors (SKPs) are multipotent dermal precursors that share similarities with neural crest stem cells (NCSCs), a shared ability for multilineage differentiation toward neural crest lineages between SKPs and NCSCs has not been fully demonstrated. Here, we report the derivation of SKPs from adult mouse skin and their directed multilineage differentiation toward neural crest lineages. Under controlled in vitro conditions, mouse SKPs were propagated and directed toward peripheral nervous system lineages such as peripheral neurons and Schwann cells, and mesenchymal lineages, such as osteogenic, chondrogenic, adipogenic, and smooth muscle cells. To ask if SKPs could generate these same lineages in vivo, a mixture of SKP-derived mesenchymal stem cells and hydroxyapatite/tricalcium phosphate was transplanted into the rat calvarial defects. Over the ensuing 4 weeks, we observed formation of osteogenic structure in the calvarial defect without any evidence of teratomas. These findings demonstrate the multipotency of adult mouse SKPs to differentiate into neural crest lineages. In addition, SKP-derived mesenchymal stem cells represent an accessible, potentially autologous source of precursor cells for tissue-engineered bone repair.