Coupled fibromodulin and SOX2 signaling as a critical regulator of metastatic outgrowth in melanoma.

We aimed to study mechanisms controlling metastatic outgrowth of melanoma into clinically relevant lesions, a critical process responsible for the majority of melanoma deaths. To this end, we developed novel in vivo models and identified molecular events that can be ascribed to their distinct phenotypes, indolent or highly metastatic. Induction of a proliferative state at distant sites was associated with high levels of the stem-like/progenitor marker, SOX2, and required the upregulation of FMOD, an extracellular matrix component, which modulates tumor-stroma interactions. Functional studies revealed a possible link between FMOD and SOX2; dual FMOD and SOX2 silencing nearly abolished brain metastasis and had a similar effect on distant metastasis to other sites. Our in vitro data suggests that FMOD and SOX2 cooperation plays an important role in tumor vasculogenic mimicry. Furthermore, we found that FMOD and SOX2 functional roles might converge at the activation of transcriptional co-factors YAP and TAZ, possibly via crosstalk with the tumor suppressor Hippo pathway. Finally, high expression of both genes in patient specimens predicted early development of brain metastasis. Thus, our study identifies FMOD and SOX2 cooperation as a novel regulatory mechanism that might be linked functionally to melanoma metastatic competence.

Extracellular matrix components and elasticity regulate mouse vaginal epithelial differentiation induced by mesenchymal cells†.

Oviduct, uterus, and vagina are derived from Müllerian ducts. But only in the vagina, the epithelium differentiates into stratified layers. Organ-specific secreted factors derived from the stroma of a neonatal mouse induce epithelial differentiation in the female reproductive tracts. However, the effects of the components and mechanical property of extracellular matrix (ECM) on the regulation of gene expression in the mesenchymal cells of neonatal stroma and differentiation of epithelium in the female reproductive tracts have been overlooked. In the present study, we have developed a simple 3D neonatal vaginal model using clonal cell lines to study the effect of ECM's components and stiffness on the epithelial stratification. Transcriptome analysis was performed by DNA-microarray to identify the components of ECM involved in the differentiation of vaginal epithelial stratification. The knockdown experiment of the candidate genes relating to vaginal epithelial stratification was focused on fibromodulin (Fmod), a collagen cross-linking protein. FMOD was essential for the expression of Bmp4, which encodes secreted factors to induce the epithelial stratification of vaginal mesenchymal cells. Furthermore, stiffer ECM as a scaffold for epithelial cells is necessary for vaginal epithelial stratification. Therefore, the components and stiffness of ECM are both crucial for the epithelial stratification in the neonatal vagina.

Influence of microcurrent on the modulation of remodelling genes in a wound healing assay.

The literature has shown the beneficial effects of microcurrent (MC) therapy on tissue repair. We investigated if the application of MC at 10 µA/90 s could modulate the expression of remodeling genes transforming growth factor beta (Tgfb), connective tissue growth factor (Ctgf), insulin-like growth factor 1 (Igf1), tenascin C (Tnc), Fibronectin (Fn1), Scleraxis (Scx), Fibromodulin (Fmod) and tenomodulin in NIH/3T3 fibroblasts in a wound healing assay. The cell migration was analyzed between days 0 and 4 in both fibroblasts (F) and fibroblasts + MC (F+MC) groups. On the 4th day, cell viability and gene expression were also analyzed after daily MC application. Higher expression of Ctgf and lower expression of Tnc and Fmod, respectively, were observed in the F+MC group in relation to F group (p < 0.05), and no difference was observed between the groups for the genes Tgfb, Fn1 and Scx. In cell migration, a higher number of cells in the scratch region was observed in group F+MC (p < 0.05) compared to group F on the 4th day, and the cell viability assay showed no difference between the groups. In conclusion, MC therapy at an intensity/time of 10 µA/90 s with 4 daily applications did not affect cell viability, stimulated fibroblasts migration with the involvement of Ctgf, and reduced the Tnc and Fmod expression.

Identification of Lumican and Fibromodulin as Hub Genes Associated with Accumulation of Extracellular Matrix in Diabetic Nephropathy.

INTRODUCTION: Diabetic nephropathy (DN) remains a major cause of end-stage renal disease. The development of novel biomarkers and early diagnosis of DN are of great clinical importance. The goal of this study was to identify hub genes with diagnostic potential for DN by weighted gene co-expression network analysis (WGCNA). METHODS: Gene Expression Omnibus database was searched for microarray data including distinct types of CKD. Gene co-expression network was constructed, and modules specific for DN were identified by WGCNA. Gene ontology (GO) analysis was performed, and the hub genes were screened out within the selected gene modules. In addition, cross-validation was performed in an independent dataset and in samples of renal biopsies with DN and other types of glomerular diseases. RESULTS: Dataset GSE99339 was selected, and a total of 179 microdissected glomeruli samples were analyzed, including DN, normal control, and 7 groups of other glomerular diseases. Twenty-three modules of the total 10,947 genes were grouped by WGCNA, and a module was specifically correlated with DN (r = 0.54, p = 9e-15). GO analysis showed that module genes were mainly enriched in the accumulation of extracellular matrix (ECM). LUM, ELN, FBLN1, MMP2, FBLN5, and FMOD were identified as hub genes. Cross verification showed LUM and FMOD were higher in the DN group and were negatively correlated with estimated glomerular filtration rate (eGFR). In renal biopsies, expression levels of LUM and FMOD were higher in DN than IgA nephropathy, membranous nephropathy, and normal controls. CONCLUSION: By using WGCNA approach, we identified LUM and FMOD related to ECM accumulation and were specific for DN. These 2 genes may represent potential candidate diagnostic biomarkers of DN.

Fibromodulin is upregulated by oxidative stress through the MAPK/AP-1 pathway to promote pancreatic stellate cell activation.

BACKGROUND/OBJECTIVES: Fibromodulin (FMOD) expression in chronic pancreatitis (CP) tissues and its effect on PSC was unknown. Our aim was to investigate the role of FMOD in regulating PSC profibrogenic phenotype and the molecular mechanism of CP. METHODS: Rat CP models were induced by dibutyltin dichloride. Pancreatic fibrosis was evaluated by Sirius Red staining. The expression of FMOD and α-SMA was measured, the correlation between FMOD expression and fibrosis was investigated in CP models and CP patients. The effects of FMOD on PSCs were examined by CCK-8 and migration assays. We investigated the mechanisms underlying FMOD expression using MND and a MAPK pathway inhibitor. Luciferase reporter and chromatin immunoprecipitation assays were used to investigate the effects of AP-1 on FMOD expression. RESULTS: Sirius Red staining revealed high collagen deposition in model rats. Higher expression of FMOD and α-SMA was observed in fibrotic tissues, and the expression of FMOD was correlated with that of α-SMA and the areas of Sirius Red staining. Upregulation of FMOD increased the expression of collagen I and α-SMA and the proliferation and migration of PSCs. MND induced FMOD and α-SMA expression, and knockdown of FMOD abated α-SMA expression. ERK and JNK inhibitors attenuated FMOD expression as induced by MND. AP-1 upregulated the expression of FMOD. AP-1 binds to the FMOD promoter and transcriptionally regulates FMOD expression. CONCLUSION: FMOD levels are upregulated in fibrosis tissues in CP and it is a critical downstream mediator of oxidative stress. FMOD induces PSC activation and maintains the fibrosis phenotype of PSCs.

[Effect of Silencing FMOD on Proliferation and Migration of H322 Cells and Its Mechanism].

OBJECTIVE: To investigate the regulation of fibromodulin (FMOD) on proliferation, adhesion and migration of non-small cell lung cancer cell line H322, and discuss its action mechanism. METHODS: H322 cells were randomly divided into control group, small interfering RNA (siRNA) silencing FMOD ( FMOD siRNA) group and control siRNA (Con siRNA) group. FMOD siRNA and Con siRNA were transfected into H322 cells. The cell viability of each group was detected by CCK-8 method. The adhesion ability of cells was detected by fluorescein diacetate (FDA) fluorescent staining. The cell migration ability was detected by Transwell method. Real time-PCR was used to detect the mRNA expressions of Cyclin D1, intercellular adhesion molecule -1 （ICAM-1）, E-cadherin, FMOD, transforming growth factor-ß (TGF-ß), Smad2, Smad3, Smad4 and Smad7 in cells. The protein expressions of Cyclin D1, ICAM-1, E-cadherin, FMOD, TGF-ß1, Smad2, Smad3, Smad4 and Smad7 were detected by Western blot. RESULTS: Compared with the Con siRNA group, the cell viability, cell adhesion and migration ability of the FMOD siRNA group were decreased, and the difference was statistically significant ( P<0.01). There was no significant difference between the control group and the Con siRNA group. Real time-PCR and Western blot results showed that the mRNA and protein expression levels of Cyclin D1, ICAM-1, TGF-ß1, Smad2, Smad3 and Smad4 were decreased in FMOD siRNA group, compared with Con siRNA group, while the mRNA and protein expression levels of E-cadherin and Smad7 are elevated. CONCLUSION: Silencing of the FMOD gene significantly reduces the proliferation, adhesion and migration of H322 cells, which may be conducted by inhibiting the TGF-ß/Smad signaling pathway.

Fibromodulin and regulation of the intricate balance between myoblast differentiation to myocytes or adipocyte-like cells.

Interactions between myoblasts and the surrounding microenvironment led us to explore the role of fibromodulin (FMOD), an extracellular matrix protein, in the maintenance of myoblast stemness and function. Microarray analysis of FMODkd myoblasts and in silico studies were used to identify the top most differentially expressed genes in FMODkd, and helped establish that FMOD-based regulations of integral membrane protein 2a and clusterin are essential components of the myogenic program. Studies in knockout, obese, and diabetic mouse models helped characterize the operation of a novel FMOD-based regulatory circuit that controls myoblast switching from a myogenic to a lipid accumulation fate. FMOD regulation of myoblasts is an essential part of the myogenic program, and it offers opportunities for the development of therapeutics for the treatment of different muscle diseases.-Lee, E. J., Jan, A. T., Baig, M. H., Ahmad, K., Malik, A., Rabbani, G., Kim, T., Lee, I.-K., Lee, Y. H., Park, S.-Y., Choi, I. Fibromodulin and regulation of the intricate balance between myoblast differentiation to myocytes or adipocyte-like cells.

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive-mechanical-loading porcine cutaneous wounds.

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule-fibromodulin (FMOD) protein-can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring-especially hypertrophic scarring.

Downregulated microRNA-340-5p promotes proliferation and inhibits apoptosis of chondrocytes in osteoarthritis mice through inhibiting the extracellular signal-regulated kinase signaling pathway by negatively targeting the FMOD gene.

PURPOSE: Osteoarthritis (OA) is a degenerative joint disease that leads to the destruction of joint function. The aim of this study is to investigate the effects of microRNA-340-5p (miR-340-5p) and its target gene, FMOD, on the proliferation and apoptosis of chondrocytes in mice with OA through the extracellular signal-regulated kinase (ERK) signaling pathway. MATERIALS: Twenty healthy C57BL/6J mice aged 15 months with a weight of 50 ± 2 g were selected. Ten mice were treated using a unilateral knee anterior cruciate ligament transection as well as a medial meniscectomy to establish the OA model. Besides, another 10 mice were used as the control group. METHODS: A reverse transcription quantitative polymerase chain reaction and Western blot analysis methods were used to examine the expressions of related genes in cells of each group. A 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide assay and flow cytometry were also conducted to evaluate the cell function after transfection had been completed. RESULTS: The expressions of fibromodulin (FMOD), type II collagen (Col II), B-cell lymphoma-2 (Bcl-2), sex-determining region of Y chromosome (SRY)-related high-mobility group-box gene 9 (Sox9), and proliferating cell nuclear antigen (PCNA) were decreased, whereas the expressions of miR-340-5p, runt-related transcription factor-2 (Runx2), Bcl-2-associated X protein (Bax), and ERK1/2 were elevated in the OA mice. Downregulation of miR-340-5p and upregulation of FMOD decreased the expressions of Runx2, Bax, and ERK1/2, and cell apoptosis of chondrocytes, and increased the expressions of FMOD, Col II, Bcl-2, Sox9, and PCNA, and cell proliferation. CONCLUSION: This study suggests that downregulation of miR-340-5p plays a role in promoting cell proliferation and suppressing cell apoptosis of chondrocytes in OA mice through inhibition of the ERK signaling pathway via the FMOD gene.

Glial Cell Line-Derived Neurotrophic Factor (GDNF) Promotes Angiogenesis through the Demethylation of the Fibromodulin (FMOD) Promoter in Glioblastoma.

BACKGROUND Angiogenesis plays an important role in the progression of glioblastoma, with a high degree of malignancy. Previous studies have proved that glial cell line-derived neurotrophic factor (GDNF) and fibromodulin (FMOD) are strongly expressed in human glioblastoma. The purpose of this study was to explore the roles of GDNF and FMOD in angiogenesis and the molecular mechanisms underlying these roles in human glioblastoma. MATERIAL AND METHODS The effects of GDNF on the expression and secretion of vascular endothelial growth factor (VEGF) in human glioblastoma cell line U251 and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated. The molecular mechanism of GDNF-induced expression of FMOD was explored. The roles of FMOD in GDNF-induced expression and secretion of VEGF and angiogenesis were also examined. RESULTS In the present study, we showed that GDNF promoted the expression and secretion of VEGF in U251 cells. VEGF mediated GDNF-induced angiogenesis in human glioblastoma. In addition, GDNF significantly upregulated the expression of FMOD in U251 cells. Mechanistically, the results of luciferase reporter assay and methylation-specific PCR (MSP) demonstrated that GDNF facilitated the demethylation of the FMOD promoter. More importantly, we found that FMOD acted as an important mediator in VEGF expression and angiogenesis induced by GDNF in human glioblastoma. CONCLUSIONS Collectively, our data show that GDNF promotes angiogenesis through demethylation of the FMOD promoter in human glioblastoma, indicating that GDNF and FMOD may be potential therapeutic targets for glioblastoma.

Fibromodulin Is Essential for Fetal-Type Scarless Cutaneous Wound Healing.

In contrast to adult and late-gestation fetal skin wounds, which heal with scar, early-gestation fetal skin wounds display a remarkable capacity to heal scarlessly. Although the underlying mechanism of this transition from fetal-type scarless healing to adult-type healing with scar has been actively investigated for decades, in utero restoration of scarless healing in late-gestation fetal wounds has not been reported. In this study, using loss- and gain-of-function rodent fetal wound models, we identified that fibromodulin (Fm) is essential for fetal-type scarless wound healing. In particular, we found that loss of Fm can eliminate the ability of early-gestation fetal rodents to heal without scar. Meanwhile, administration of fibromodulin protein (FM) alone was capable of restoring scarless healing in late-gestation rat fetal wounds, which naturally heal with scar, as characterized by dermal appendage restoration and organized collagen architectures that were virtually indistinguishable from those in age-matched unwounded skin. High Fm levels correlated with decreased transforming growth factor (TGF)-ß1 expression and scarless repair, while low Fm levels correlated with increased TGF-ß1 expression and scar formation. This study represents the first successful in utero attempt to induce scarless repair in late-gestation fetal wounds by using a single protein, Fm, and highlights the crucial role that the FM-TGF-ß1 nexus plays in fetal-type scarless skin repair.

Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

Differentiation of muscle satellite cells (MSCs) involves interaction of the proteins present in the extracellular matrix (ECM) with MSCs to regulate their activity, and therefore phenotype. Herein, we report fibromodulin (FMOD), a member of the proteoglycan family participating in the assembly of ECM, as a novel regulator of myostatin (MSTN) during myoblast differentiation. In addition to having a pronounced effect on the expression of myogenic marker genes [myogenin (MYOG) and myosin light chain 2 (MYL2)], FMOD was found to maintain the transcriptional activity of MSTN Moreover, coimmunoprecipitation and in silico studies performed to investigate the interaction of FMOD helped confirm that it antagonizes MSTN function by distorting its folding and preventing its binding to activin receptor type IIB. Furthermore, in vivo studies revealed that FMOD plays an active role in healing by increasing satellite cell recruitment to sites of injury. Together, these findings disclose a hitherto unrecognized regulatory role for FMOD in MSCs and highlight new mechanisms whereby FMOD circumvents the inhibitory effects of MSTN and triggers myoblast differentiation. These findings offer a basis for the design of novel MSTN inhibitors that promote muscle regeneration after injury or for the development of pharmaceutical agents for the treatment of different muscle atrophies.-Lee, E. J., Jan, A. T., Baig, M. H., Ashraf, J. M., Nahm, S.-S., Kim, Y.-W., Park, S.-Y., Choi, I. Fibromodulin: a master regulator of myostatin controlling progression of satellite cells through a myogenic program.

Development of myotendinous-like junctions that anchor cardiac valves requires fibromodulin and lumican.

BACKGROUND: There are many patients that exhibit connective tissue related cardiac malformations but do not have mutations in collagen genes. The Small Leucine Rich Proteoglycans (SLRP) fibromodulin (FMOD) and lumican (LUM) bind collagen and regulate fibril assembly in other biological contexts. RESULTS: FMOD deficient mice and double deficient FMOD; LUM mice exhibited anomalies in regions where cardiac valve tissue interdigitates with adjacent muscle for support. Ectopic connective and/or myocardial tissue(s) was associated with the more severe cardiac valve anomalies in FMOD; LUM deficient mice. At postnatal day 0 (P0) there was an increase in the mesenchymal cell number in the regions where valve cusps anchor in FMOD; LUM deficient mice compared to WT. The cardiac valve anomalies correlated with the highest levels of FMOD expression in the heart and also where myotendinous junctions (MTJ) components biglycan, collagen type I alpha 1, and collagen type VI, are also localized. CONCLUSIONS: The postnatal assembly of the collagen-rich ECM in regions where cardiac valves anchor, that we have designated 'myotendinous-like junctions' (MTLJ) requires the SLRPs FMOD and LUM. Moreover, FMOD and LUM may facilitate mesenchymal cell differentiation in late stages of cardiac valve development. Developmental Dynamics 245:1029-1042, 2016. © 2016 Wiley Periodicals, Inc.

FMOD Alleviates Depression-Like Behaviors by Targeting the PI3K/AKT/mTOR Signaling After Traumatic Brain Injury.

Depression frequently occurs following traumatic brain injury (TBI). However, the role of Fibromodulin (FMOD) in TBI-related depression is not yet clear. Previous studies have suggested FMOD as a potential key factor in TBI, yet its association with depression post-TBI and underlying mechanisms are not well understood. Serum levels of FMOD were measured in patients with traumatic brain injury using qPCR. The severity of depression was assessed using the self-depression scale (SDS). Neurological function, depressive state, and cognitive function in mice were assessed using the modified Neurological Severity Score (mNSS), forced swimming test (FST), tail suspension test (TST), Sucrose Preference Test (SPT), and morris water maze (MWM). The morphological features of mouse hippocampal synapses and neuronal dendritic spines were revealed through immunofluorescence, transmission electron microscopy, and Golgi-Cox staining. The protein expression levels of FMOD, MAP2, SYP, and PSD95, as well as the phosphorylation levels of the PI3K/AKT/mTOR signaling pathway, were detected through Western blotting. FMOD levels were decreased in TBI patients' serum. Overexpression of FMOD preserved neuronal function and alleviated depression-like behaviour, increased synaptic protein expression, and induced ultrastructural changes in hippocampal neurons. The increased phosphorylation of PI3K, AKT, and mTOR suggested the involvement of the PI3K/AKT/mTOR signaling pathway in FMOD's protective effects. FMOD exhibits potential as a therapeutic target for depression related to TBI, with its protective effects potentially mediated through the PI3K/AKT/mTOR signaling pathway.

Downregulation of fibromodulin attenuates inflammatory signaling and atrial fibrosis in spontaneously hypertensive rats with atrial fibrillation via inhibiting TLR4/NLRP3 signaling pathway.

BACKGROUND: Myocardial fibrosis is an important factor in the induction and maintenance of atrial fibrillation (AF). Fibromodulin (FMOD) promotes fibrotic gene expression. However, its specific role in spontaneously hypertensive rats (SHR)-AF remains unclear. METHODS: We analyzed FMOD mRNA and protein expression in rat atrial tissues using RT-qPCR, Western blot analysis, and immunohistochemistry. Histopathological examination of atrial tissues was performed using hematoxylin and eosin (H&E), Masson's trichrome, and Picrosirius red staining. The levels of inflammatory and fibrosis-related proteins were measured using Western blot analysis. RESULTS: FMOD relative mRNA and protein expression levels were notably upregulated in atrial tissues of both AF groups (normal-AF and SHR-AF groups) than that in atrial tissues of the no-AF group (normal and SHR group). This effect was particularly pronounced in the SHR-AF group. Pathological changes revealed that the extracellular matrix, collagen, collagen fibers, and left atrial diameter were notably increased in the atrial tissues from the SHR-AF group compared to those in the atrial tissues from the SHR group, whereas the left ventricular fractional shortening and left ventricular ejection fraction were notably lower. Expression of TLR4, MyD88, NLRP3, TGF-ß1, collagen I, and collagen II mRNA were clearly higher in atrial tissues from the SHR-AF group than in those from the SHR group. Protein levels of TLR4, MyD88, NLRP3, Cleavage-Caspase-1, Cleavage-IL-1ß, TGF-ß1, p-Smad2, collagen I, and collagen II were clearly higher in atrial tissues from the SHR-AF group than in those from the SHR group. FMOD knockdown inhibited atrial fibrosis, collagen accumulation, and the TLR4/MyD88/NLRP3 signaling pathway. CONCLUSION: Downregulation of FMOD attenuated inflammatory signaling and atrial fibrosis in SHR-AF by inhibiting the TLR4/NLRP3 signaling pathway. Therefore, FMOD may be a promising therapeutic target in AF.

Fibromodulin Gene Variants (FMOD) as Potential Biomarkers for Prostate Cancer and Benign Prostatic Hyperplasia.

By the year 2050, the world's elderly population may increase exponentially, raising the rate of disease characteristic of this group, such as prostate cancer (PCa) and benign prostatic hyperplasia (BPH). Prostate disorders have a multifactorial etiology, especially age and genetic factors. Currently, PCa is the second most frequent neoplasm in the male population worldwide. The fibromodulin gene encodes a small leucine-rich proteoglycan (SLRP) which acts in the collagen fibrillogenesis pathway, cell adhesion, and modulation of TGF-ß signaling pathways, which has been recently associated with PCa. The present study sequenced the coding region of the FMOD in a sample of 44 PCa, 90 BPH, and 82 controls from a Brazilian population, and the results identified 6 variants: 2 missenses (p.(Tyr42Ser) and p.(Pro24Ala)); 3 synonymous (p.(His253=), p.(Asn353=), and p.(Glu79=)); and 1 intronic (c.980-114A>G). Of these, p.(Tyr42Ser), p.(Pro24Ala), and p.(Asn353=) are rare variants, and p.(Tyr42Ser) was predicted as potential pathogenic by the algorithms used here, in addition to not being observed in controls, suggesting that may be a potential biomarker for development of PCa and BPH. In conclusion, we identified for the first time, in Brazilian individuals with PCa and BPH, a potentially pathogenic variant in the analysis of FMOD gene. Further studies are needed to investigate the deleterious effect of this variant on the structure and/or function of the FMOD protein.

A-kinase anchoring protein 13 interacts with the vitamin D receptor to alter vitamin D-dependent gene activation in uterine leiomyoma cells.

OBJECTIVE: To determine if A-kinase anchoring protein 13 (AKAP13) interacts with the vitamin D receptor (VDR) to alter vitamin D-dependent signaling in fibroid cells. Uterine leiomyomas (fibroids) are characterized by a fibrotic extracellular matrix and are associated with vitamin D deficiency. Treatment with vitamin D (1,25-dihydroxyvitamin D3) reduces fibroid growth and extracellular matrix gene expression. A-kinase anchoring protein 13 is overexpressed in fibroids and interacts with nuclear hormone receptors, but it is not known whether AKAP13 may interact with the VDR to affect vitamin D signaling in fibroids. DESIGN: Laboratory studies. SETTING: Translational science laboratory. INTERVENTION(S): Human immortalized fibroid or myometrial cells were treated with 1,25-hydroxyvitamin D3 (1,25(OH)2D3) and transfected using expression constructs for AKAP13 or AKAP13 mutants, RhoQL, C3 transferase, or small interfering ribonucleic acids (RNAs). MAIN OUTCOME MEASURE(S): Messenger ribonucleic acid (mRNA) levels of AKAP13, fibromodulin, and versican as measured by quantitative real-time polymerase chain reaction. Glutathione S-transferase-binding assays. Vitamin D-dependent gene activation as measured by luciferase assays. RESULT(S): 1,25(OH)2D3 resulted in a significant reduction in mRNA levels encoding AKAP13, versican, and fibromodulin. Small interfering RNA silencing of AKAP13 decreased both fibromodulin and versican mRNA levels. Glutathione S-transferase-binding assays revealed that AKAP13 bound to the VDR through its nuclear receptor interacting region. Cotransfection of AKAP13 and VDR significantly reduced vitamin D-dependent gene activation. RhoA pathway inhibition partially relieved repression of vitamin D-dependent gene activation by AKAP13. CONCLUSION(S): These data suggest that AKAP13 inhibited the vitamin D receptor activation by a mechanism that required, at least in part, RhoA activation.

Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Interaction of Fibromodulin and Myostatin to Regulate Skeletal Muscle Aging: An Opposite Regulation in Muscle Aging, Diabetes, and Intracellular Lipid Accumulation.

The objective of this study was to investigate fibromodulin (FMOD) and myostatin (MSTN) gene expressions during skeletal muscle aging and to understand their involvements in this process. The expressions of genes related to muscle aging (Atrogin 1 and Glb1), diabetes (RAGE and CD163), and lipid accumulation (CD36 and PPARγ) and those of FMOD and MSTN were examined in CTX-injected, aged, MSTN-/-, and high-fat diet (HFD) mice and in C2C12 myoblasts treated with ceramide or grown under adipogenic conditions. Results from CTX-injected mice and gene knockdown experiments in C2C12 cells suggested the involvement of FMOD during muscle regeneration and myoblast proliferation and differentiation. Downregulation of the FMOD gene in MSTN-/- mice, and MSTN upregulation and FMOD downregulation in FMOD and MSTN knockdown C2C12 cells, respectively, during their differentiation, suggested FMOD negatively regulates MSTN gene expression, and MSTN positively regulates FMOD gene expression. The results of our in vivo and in vitro experiments indicate FMOD inhibits muscle aging by negatively regulating MSTN gene expression or by suppressing the action of MSTN protein, and that MSTN promotes muscle aging by positively regulating the expressions of Atrogin1, CD36, and PPARγ genes in muscle.

Canonical and noncanonical TGF-ß signaling regulate fibrous tissue differentiation in the axial skeleton.

Previously, we showed that embryonic deletion of TGF-ß type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-ß regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-ß stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-ß did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-ß-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-ß-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-ß treatment. Next, the role of several noncanonical TGF-ß pathways were tested. We found that ERK1/2 was activated by TGF-ß and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-ß regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFß-mediated fibrous differentiation in sclerotome.