Calponin H1 Research Articles

Inhibiting Monoacylglycerol Lipase Suppresses RANKL-Induced Osteoclastogenesis and Alleviates Ovariectomy-Induced Bone Loss.

Osteoporosis is a common chronic metabolic bone disease characterized by reduced trabecular bone and increased bone fragility. Monoacylglycerol lipase (MAGL) is a lipolytic enzyme to catalyze the hydrolysis of monoglycerides and specifically degrades the 2-arachidonoyl glycerol (2-AG). Previous studies have identified that 2-AG is the mainly source for arachidonic acid and the most abundant endogenous agonist of cannabinoid receptors. Considering the close relationship between inflammatory mediators/cannabinoid receptors and bone metabolism, we speculated that MAGL may play a role in the osteoclast differentiation. In the present study, we found that MAGL protein expression increased during osteoclast differentiation. MAGL knockdown by adenovirus-mediated shRNA in bone marrow-derived macrophages demonstrated the suppressive effects of MAGL on osteoclast formation and bone resorption. In addition, pharmacological inhibition of MAGL by JZL184 suppressed osteoclast differentiation, bone resorption, and osteoclast-specific gene expression. Activation of the Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways was inhibited by JZL184 and deletion of MAGL. Our in vivo study indicated that JZL184 ameliorated bone loss in an ovariectomized mouse model. Furthermore, overexpressing H1 calponin partially alleviated the inhibition caused by JZL184 or MAGL deletion on osteoclastogenesis. Therefore, we conclude that targeting MAGL may be a novel therapeutic strategy for osteoporosis.

Potential diagnostic biomarkers: differential expression of LMP2/β1i and cyclin B1 in human uterine leiomyosarcoma.

Whilst most uterine smooth muscle neoplasms are benign, uterine leiomyosarcoma (Ut-LMS) is extremely malignant with a high incidence of metastasis and recurrence. Gynecological tumors are often associated with female hormone secretion, but no strong link has been detected between human Ut-LMS and the hormonal environment. In fact, the risk factors for Ut-LMS are poorly understood. In addition, no diagnostic biomarkers for differentiating between leiomyoma, a benign tumor, and malignant Ut-LMS have been found. Interestingly, mice that were homozygously deficient for LMP2/β1i were found to spontaneously develop Ut-LMS and exhibited a Ut-LMS prevalence of ~40% by 14 months of age. Thus, analyzing potential risk factors for Ut-LMS (such as LMP2/β1i) might aid the development of diagnostic biomarkers and clinical treatments for the condition. Fifty-seven patients (age range: 32-83 years) who had been diagnosed with uterine mesenchymal tumors were chosen from a pathological archive. Tissue samples from these patients were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin for standard histological examination or were subjected to further processing for immunohistochemical (IHC) examination. Serial Ut-LMS, bizarre leiomyoma, leiomyoma, and myometrium sections were subjected to IHC staining of β-smooth muscle actin, estrogen receptor, cyclin B1, LMP2/β1i, calponin h1, ki-67, tumor protein p53, and progesterone receptor. The Ut-LMS samples were positive for cyclin B1 and negative for LMP2/β1i, while the opposite result was obtained for bizarre leiomyoma, leiomyoma, and myometrium samples. The expression pattern of LMP2/β1i and cyclin B1 might be a diagnostic biomarker for human Ut-LMS. Studies of the biological roles of LMP2/β1i and/or cyclin B1 could lead to the elucidation of new targets for therapies against Ut-LMS.

Loss of calponin h1 confers anoikis resistance and tumor progression in the development of high-grade serous carcinoma originating from the fallopian tube epithelium.

Increasing evidence indicates that ovarian high-grade serous carcinoma (HGSC) originates from the fallopian tube epithelium and metastasizes to the ovary as the secondary site. A working hypothesis is that detached tubal HGSC cells survive anoikis and implant on the ovary. In this study, we found that downregulation of calponin h1 (CNN1) is necessary for the anoikis survival and cell transformation. CNN1 was progressively downregulated in cells and tissues representing different stages of HGSC development from fallopian tube epithelium (FTE). Knock down of CNN1 in immortalized human FTE cells conferred gains of resistance to anoikis and transformation phenotypes including anchorage independent growth (AIG) and xenograft tumorigenesis in NSG mice. Conversely, overexpression of CNN1 in RAS-transformed FTE cells resulted in an almost complete loss of AIG and tumorigenesis. Besides, there was a dramatic change of cell morphology from a polygonal, raised appearance to a round and flattened one. Increase in cell adhesion to laminin and collagen, and reduction in cell motility, anoikis resistance and invasiveness were also observed. A microarray analysis revealed upregulation of genes involved in cytoskeleton stabilization and signal transduction, and downregulation of genes involved in cytokine and chemokine activities. The study disclosed multiple tumor suppressor roles of CNN1 in the development of HGSC from the fallopian tube, and loss of CNN1 expression is crucial for its metastasis to a new site.

Characterization of Leiomyomatoid Angiomatous Neuroendocrine Tumour (LANT)-like Tumour in the Myometrium with Histopathological Examination.

Leiomyomatoid angiomatous neuroendocrine tumour (LANT) is possibly a new disease entity that was reported as a dimorphic neurosecretory tumour with a leiomyomatous vascular component; it was found in the pituitary. We describe uterine LANT-like malignant tumour in a 45-year-old woman with uterine mesenchymal tumour, diagnosed clinically as uterine leiomyoma. She underwent laparoscopic myomectomy. The tumour consisted of hyalinized vasculature, containing factor VIII-positive endothelium and α-smooth muscle actin-positive vascular smooth muscle cells, and stromal cells, expressing neuroadhesion molecules. Both vascular and stromal components diffusely expressed chromogranin A. Histopathological examinations of uterine LANT-like malignant tumour revealed the common characteristic abnormalities of malignant uterine mesenchymal tumours, i.e. leiomyosarcomas. From our research, defective expression of calponin H1 and proteasome β9 (PSMB9)/β1i is observed in uterine LANT-like malignant tumour similarly to immunopathological findings of uterine leiomyosarcoma. These findings meet the definition of uterine LANT-like malignant tumour, and the research findings of our clinical case suggest that LANT is a special type of neuroendocrine neoplasm and is not organ specific.

XBP 1-Deficiency Abrogates Neointimal Lesion of Injured Vessels Via Cross Talk With the PDGF Signaling.

Smooth muscle cell (SMC) migration and proliferation play an essential role in neointimal formation after vascular injury. In this study, we intended to investigate whether the X-box-binding protein 1 (XBP1) was involved in these processes. In vivo studies on femoral artery injury models revealed that vascular injury triggered an immediate upregulation of XBP1 expression and splicing in vascular SMCs and that XBP1 deficiency in SMCs significantly abrogated neointimal formation in the injured vessels. In vitro studies indicated that platelet-derived growth factor-BB triggered XBP1 splicing in SMCs via the interaction between platelet-derived growth factor receptor β and the inositol-requiring enzyme 1α. The spliced XBP1 (XBP1s) increased SMC migration via PI3K/Akt activation and proliferation via downregulating calponin h1 (CNN1). XBP1s directed the transcription of mir-1274B that targeted CNN1 mRNA degradation. Proteomic analysis of culture media revealed that XBP1s decreased transforming growth factor (TGF)-β family proteins secretion via transcriptional suppression. TGF-β3 but not TGF-β1 or TGF-β2 attenuated XBP1s-induced CNN1 decrease and SMC proliferation. This study demonstrates for the first time that XBP1 is crucial for SMC proliferation via modulating the platelet-derived growth factor/TGF-β pathways, leading to neointimal formation.

Evaluation of myofibroblasts in oral squamous cell carcinoma using H1 calponin: An immunohistochemical study.

Background:Oral squamous cell carcinoma (OSCC) is the most common malignancy of the oral mucosa. Stromal myofibroblasts play an important role in tumor invasion and metastasis, due to its ability to modify the extracellular matrix. The purpose of this study was to evaluate and compare the presence of myofibroblasts in normal mucosa, early invasive carcinoma and different grades of OSCC.Materials and Methods:The study included the archival tissues of 18 OSCC of well, moderate and poorly differentiated grades, three early invasive carcinomas and five normal mucosa. Myofibroblasts were identified by immunohistochemical detection of h1 calponin.Results:The percentage and intensity of h1 calponin were examined and positive immunostaining was observed in the myofibroblasts of all SCCs and early invasive carcinomas; however, these cells did not stain in the normal epithelium specimens. The presence of myofibroblasts was significantly higher in invasive pattern of OSCCs compared to normal mucosa cases (P < 0.070). A significant difference was not observed between the different grades of OSCC (P ≤ 0.812).Conclusion:These findings show the presence of myofibroblasts in OSCC but not in normal mucosa, suggesting that the genetically altered epithelium (carcinomatous epithelium) may have an inductive effect on the adjacent stroma to produce myofibroblasts. Also transdifferentiation of myofibroblasts is induced somewhere in the invasive stage of SCC irrespective of the epithelial cell differentiation.

Altered vascular smooth muscle cell differentiation in the endometrial vasculature in menorrhagia.

How does the smooth muscle content and differentiation stage of vascular smooth muscle cells (VSMCs) in endometrial blood vessels change according to the different phases of the menstrual cycle and is this altered in women with menorrhagia? The smooth muscle content (as a proportion of the vascular cross-sectional area) of endometrial blood vessels remained unchanged during the normal menstrual cycle and in menorrhagia; however, expression of the VSMC differentiation markers, smoothelin and calponin, was dysregulated in endometrial blood vessels in samples from women with menorrhagia compared with controls. Menorrhagia affects 30% of women of reproductive age and is the leading indication for hysterectomy. Previous studies have suggested important structural and functional roles for endometrial blood vessels, including impaired vascular contractility. Differentiation of VSMC from a synthetic to contractile state is associated with altered cellular phenotype that contributes to normal blood flow and pressure. This vascular maturation process has been little studied in endometrium both across the normal menstrual cycle and in menorrhagia. Endometrial biopsies were taken from hysterectomy specimens or by pipelle biopsy prior to hysterectomy in controls without endometrial pathology and in women with menorrhagia (n = 7 for each of proliferative, early-secretory, mid-secretory and late-secretory phases for both groups). Biopsies were formalin fixed and embedded in paraffin wax. Paraffin-embedded sections were immunostained for α smooth muscle actin (αSMA), myosin heavy chain (MyHC), H-caldesmon, desmin, smoothelin and calponin (h1 or basic). VSMC content was measured in 25 αSMA(+) vascular cross sections per sample and expressed as a ratio of the muscular area:gross vascular cross-sectional area. VSMC differentiation was analysed by the presence/absence of differentiation markers compared with αSMA expression. Smoothelin and calponin expression was also analysed in relation to total number of blood vessels by double immunostaining for endothelial cell markers. Study of VSMC differentiation markers revealed decreased expression of calponin both in αSMA(+) vessels (P = 0.008) and in relation to total number of vessels (P = 0.001) in late secretory phase endometrium in menorrhagia compared with controls. Smoothelin expression in αSMA(+) vessels was increased (P = 0.03) in menorrhagia, although this was not significant in relation to the total number of vessels. In normal endometrium, the proportion of blood vessels expressing αSMA increased from 63% in proliferative endometrium to 81% in the late secretory phase (P = 0.002). The overall arterial muscle content did not differ between control and menorrhagia at any phase of the menstrual cycle, occupying 78-81% of gross vascular cross-sectional area during the different menstrual cycle phases. This study included both straight and spiral arterioles and analysed only stratum functionalis. The VSMC differentiation with respect to αSMA expression is an observational study and the data are presented as presence or absence of the differentiation markers in each field of view, corresponding with the vascular cross sections included in the study of vascular muscle content. Smoothelin and calponin have been widely implicated as important regulators of vascular tone, vascular contractility and rate of blood flow. Our results have uncovered a disparate pattern of calponin expression, potentially indicating a dysfunctional contraction mechanism in the endometrial blood vessels in menorrhagia, thus implicating calponin as a potential therapeutic target. This study was funded by Wellbeing of Women (RG1342) and Newcastle University. There are no competing interests to declare. Not applicable.

Abstract 357: The Role of X-Box Binding Protein 1 Splicing in Smooth Muscle Cell Proliferation and Neointima Formation

Rationale: Smooth muscle cell (SMC) proliferation plays an important role in neointima formation in response to vascular injury. However, the underlying mechanism is still not well understood. As one of the key transducer in endoplasmic reticulum stress response, the X-box binding protein 1 (XBP1) has been demonstrated to contribute to endothelial cell proliferation, autophagic response and apoptosis, but less is known about its role in SMC growth. Methods and Results: In this study, we intend to study the role of XBP1 in SMC proliferation and its contribution to neointima formation in response to vascular injury. Platelet derived growth factor (PDGF) induced XBP1 splicing in SMCs via the interaction between PDGF receptor and inositol requiring enzyme 1 alpha (IRE1α). Transient over-expression of the spliced XBP1 (XBP1s) increased SMC proliferation, while knockdown of XBP1 or IRE1α abolished PDGF-induced SMC proliferation. XBP1s down-regulated calponin H1 gene expression at mRNA level through microRNAs targeting the 3’-terminal untranslated region. Reconstitution of calponin H1 via adenoviral transfer could ablate XBP1s-induced SMC proliferation. Proteomics analysis of secreted proteins revealed that over-expression of XBP1s in SMCs decreased the secretion of transforming growth factor β family. Addition of exogenous TGFβ3 abolished XBP1s-induced calponin H1 suppression and SMC proliferation. High levels of TGFβ3 and calponin H1 were detected in SMCs isolated from SM22-Cre/XBP1loxP mice. These SMCs showed retardation in proliferation. Importantly, in the femoral artery injury model, XBP1 deficiency in SMCs prevented the neointima formation. Conclusion: XBP1 splicing plays an important role in SMC proliferation via regulating calponin H1 expression. Targeting XBP1 splicing in SMC may provide a novel therapeutic strategy to intervene neointima formation.

Proteasome LMP2/β1i subunit as biomarker for human uterine leiomyosarcoma

Uterine leiomyosarcoma (Ut-LMS) develops more frequently in the myometrium of the uterine body than in the uterine cervix. Although the development of gynecological tumors is often correlated with the secretion of female hormones that of Ut-LMS does not, and its risk factor(s) remain unknown. Importantly, a diagnostic biomarker that can distinguish malignant tumor Ut-LMS from benign tumor leiomyoma (LMA), has yet to be established. Therefore, the risk factor(s) associated with Ut-LMS need to be examined in order to establish a diagnosis and clinical treatment method. Mice with a homozygous deficiency for the proteasome b-ring subunit, low-molecular mass polypeptide (LMP)2/b1i spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. In recent studies, we showed that LMP2/b1i expression was absent in human Ut-LMS, but present in other human uterine mesenchymal tumors including uterine LMA. Moreover, LMP2/b1i is also known to negatively regulate human Ut-LMS tumorigenesis. Additional experiments furthermore revealed the differential expression of cyclin E and calponin h1 in human uterine mesenchymal tumors. Therefore, LMP2/b1i is a potential diagnostic biomarker when combined with the candidate molecules, cyclin E and calponin h1 for human Ut-LMS, and may be a targeted molecule for a new therapeutic approach. --------------------------------------------- Cite this article as : Hayashi T, Horiuchi A Aburatani H, Ishiko O, Yaegashi N, Kanai Y, Zharhary D, Tonegawa S, Konishi I. Proteasome LMP2/s1i subunit as biomarker for human uterine leiomyosarcoma. Int J Cancer Ther Oncol 2014; 2 (1):02018. DOI: http://dx.doi.org/10.14319/ijcto.0201.8

LMP2/b1i as a potential biomarker of human uterine mesenchymal tumors under the combination of candidate molecules, cyclin E and calponin h1

Uterine leiomyosarcoma (Ut-LMS) develops more often in myometrium of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of Ut-LMS is not substantially correlated with hormonal conditions, and the risk factor(s) are not yet known. Importantly, a diagnostic-biomarker, which distinguishes malignant tumor Ut-LMS from benign tumor leiomyoma (LMA), is yet to be established. Accordingly, it is necessary to examine risk factor(s) associated with Ut-LMS, to establish a diagnosis and a clinical treatment method. The mice with a homozygous deficiency for proteasome b-ring subunit, low-molecular mass polypeptide (LMP)2/b1i spontaneously develop Ut-LMS, with a disease prevalence of ~40% by 14 months of age. In the recent study, we found LMP2/b1i expression to be absent in human Ut-LMS, but present in other human uterine mesenchymal tumors including uterine LMA. Moreover, LMP2/b1i is reported to negatively regulate human Ut-LMS tumorigenesis. Additional experiments furthermore indicate the differential expressions of cyclin E and calponin h1 during human uterine mesenchymal tumors. Therefore, LMP2/b1i is a potential diagnostic biomarker under the combination of candidate molecules, cyclin E and calponin h1 for human Ut-LMS, and may be a targeted-molecule for a new therapeutic approach.

Abstract 18000: The Role of X-box Binding Protein 1 Splicing in Smooth Muscle Cell Proliferation and Neointima Formation

Background: Smooth muscle cell (SMC) proliferation plays an important role in neointima formation in response to vascular injury. However, the underlying mechanism is still not well understood. It has been well documented that endoplasmic reticulum (ER) stress plays a provoking role in cardiovascular disease including neointima formation. As one of the key transducer in ER stress response, the X-box binding protein 1 (XBP1) has been demonstrated to contribute to endothelial cell proliferation, autophagic response and apoptosis, but less is known about its role in SMC growth. Methods and Results: In this study, we intend to study the role of XBP1 in SMC proliferation and its contribution to neointima formation in response to vascular injury. Quantitative RT-PCR and immunohistological staining analysis revealed that XBP1 splicing was up-regulated in the arterial vasculature in aged ApoE deficient mice and in response to vascular injury in wild type mice with elevated XBP1 protein in the neointima. Platelet derived growth factor (PDGF) induced XBP1 splicing via the interaction between PDGF receptor and inositol requiring enzyme 1 alpha (IRE1α). Transient over-expression of the spliced XBP1 (XBP1s) increased SMC proliferation, while knockdown of XBP1 or IRE1α abolished PDGF-induced SMC proliferation. XBP1s down-regulated calponin H1 gene expression at mRNA and protein levels, which might be through the down-regulation of TGFβ3 signalling. Reconstitution of calponin H1 via adenoviral transfer could ablate XBP1s-induced SMC proliferation. Addition of exogenous TGFβ3 abolished XBP1s-induced calponin H1 suppression and SMC proliferation. High levels of TGFβ3 and calponin H1 were detected in SMCs isolated from SM22-Cre/XBP1 loxP mice. These SMCs showed retardation in proliferation as compared to SMCs from wild type litters. Importantly, in the femoral artery injury model, XBP1 deficiency in SMCs prevented the neointima formation. CONCLUSION: XBP1 splicing plays an important role in SMC proliferation via regulating calponin H1 expression. Targeting XBP1 splicing in SMC may provide a novel therapeutic strategy to intervene neointima formation.

Candidate Molecules and ki-67/MIB1 as Novel Diagnostic Biomarker for Human Uterine Mesenchymal Tumors

Human uterine leiomyosarcoma (LMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker, which distinguishes malignant uterine LMS from benign tumor leiomyoma (LMA), is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS, to establish a clinical treatment method. Protea some β-ring subunit LMP2/β1i-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40% by 14 months of age. We found LMP2/β1i expression to be absent in human uterine LMS, but present in human LMA. Therefore, defective-LMP2/β1i expression may be one of the risk factors for human uterine LMS. LMP2/β1i is a potential diagnostic-biomarker under the combination of candidate molecules, for instance cyclin B1, cyclin E and calponin h1 and ki-67/MIB1 counts for uterine mesenchymal tumors, especially human uterine LMS, and may be a targeted-molecule for a new therapeutic approach.

Overexpression of H1 calponin in osteoblast lineage cells leads to a decrease in bone mass by disrupting osteoblast function and promoting osteoclast formation

H1 calponin (CNN1) is known as a smooth muscle-specific, actin-binding protein which regulates smooth muscle contractive activity. Although previous studies have shown that CNN1 has effect on bone, the mechanism is not well defined. To investigate the role of CNN1 in maintaining bone homeostasis, we generated transgenic mice overexpressing Cnn1 under the control of the osteoblast-specific 3.6-kb Col1a1 promoter. Col1a1-Cnn1 transgenic mice showed delayed bone formation at embryonic stage and decreased bone mass at adult stage. Morphology analyses showed reduced trabecular number, thickness and defects in bone formation. The proliferation and migration of osteoblasts were decreased in Col1a1-Cnn1 mice due to alterations in cytoskeleton. The early osteoblast differentiation of Col1a1-Cnn1 mice was increased, but the late stage differentiation and mineralization of osteoblasts derived from Col1a1-Cnn1 mice were significantly decreased. In addition to impaired bone formation, the decreased bone mass was also associated with enhanced osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) staining revealed increased osteoclast numbers in tibias of 2-month-old Col1a1-Cnn1 mice, and increased numbers of osteoclasts co-cultured with Col1a1-Cnn1 osteoblasts. The ratio of RANKL to OPG was significantly increased in Col1a1-Cnn1 osteoblasts. These findings reveal a novel function of CNN1 in maintaining bone homeostasis by coupling bone formation to bone resorption.

Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: Morphological significance of calponin h1

Uterine leiomyosarcoma (LMS) is a highly metastatic smooth muscle neoplasm for which calponin h1 is suspected to have a biological role as a tumor-suppressor. We earlier reported that LMP2-null mice spontaneously develop uterine LMS through malignant transformation of the myometrium, thus implicating this protein as an anti-tumorigenic candidate as well. In the present study, we show that LMP2 may negatively regulate LMS independently of its role in the proteasome. Moreover, several lines of evidence indicate that although calponin h1 does not directly influence tumorigenesis, it clearly affects LMP2-induced cellular morphological changes. Modulation of LMP2 may lead to new therapeutic approaches in human uterine LMS.

Phosphorylation of h1 Calponin by PKC epsilon may contribute to facilitate the contraction of uterine myometrium in mice during pregnancy and labor

BackgroundThe timely onset of powerful uterine contractions during parturition occurs through thick and thin filament interactions, similar to other smooth muscle tissues. Calponin is one of the thin filament proteins. Phosphorylation of calponin induced by PKC-epsilon can promote the contraction of vascular smooth muscle. While the mechanism by which calponin regulates the contraction of pregnant myometrium has rarely been explored. Here, we explore whether PKC-epsilon/h1 calponin pathway contribute to regulation of myometrial contractility and development of parturition.MethodsWe detected the expression of h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon in the different stages of mice during pregnancy and in labor by the method of western blot and recorded the contraction activity of myometrium strips at the 19th day during pregnancy with different treatments by the organ bath experiments.ResultsThe level of the four proteins including h1 calponin, phosphorylated h1 calponin, PKC-epsilon and phosphorylated PKC-epsilon was significantly increased in pregnant mice myometrium as compared with that in nonpregnant mice. The ratios of phosphorylated h1 calponin/h1 calponin and phosphorylated PKC-epsilon/PKC-epsilon were reached the peak after the onset of labor in myometrium in the mice. After the treatment of more than 10(9-) mol/L Psi-RACK (PKC-epsilon activator), the contractility of myometrium strips from mice was reinforced and the level of phosphorylated h1 calponin increased at the same time which could be interrupted by the specific inhibitor of PKC-epsilon. Meanwhile, the change of the ratio of phosphorylated h1 calponin/h1 calponin was consistent with that of contraction force of mice myometrium strips.ConclusionsThese data suggest that in mice myometrium, phosphorylation of h1 calponin induced by the PKC-epsilon might facilitate the contraction of uterine in labor and regulate pregnant myometrial contractility.

Tail-Suspended Mice Lacking Calponin H1 Experience Decreased Bone Loss

Calponin h1 (CNh1) is an actin-binding protein originally isolated from vascular smooth muscle and has been reported to suppress bone formation. We are therefore curious how CNh1 is involved in bone loss that is caused by space flight in microgravity. We assessed the effects of tail suspension (TS) in C57BL/6J wild (CN+/+) and CNh1-deleted (CN-/-) mice to elucidate the role of CNh1 in bone loss under weightless conditions. Bone mineral density (BMD) of tibiae was measured by single energy X-ray absorptiometry, and bone volume fraction (BV/TV), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were measured by bone histomorphometry. BMD, BV/TV, MAR, and BFR/BS were lower in CN+/+ mice with TS than in those without. In the CN-/- group, however, the decrease in each of these parameters by TS was ameliorated. Decreases in serum osteocalcin levels by TS in CN+/+ mice were attenuated in CN-/- mice. Furthermore, urinary deoxypyridinolin (DPD), an indicator of bone resorption, was increased in CN+/+ mice following TS, but not in CN-/- mice. In transfection experiments, the degree of induction of bone formation markers, alkaline phosphatase (ALP) activity and bone morphogenetic protein (BMP)-4 mRNA expression, under stimulation with BMP-2, was lower in MC3T3-E1 mouse osteoblast-like cells expressing CNh1 than that in mock transfected cells. Notably, the BMP-2-induced ALP activity was decreased by CNh1 expression, which was partially rescued by treatment with the Rho kinase inhibitor Y27632. Taken together, these results indicate that CNh1 is responsible for weightlessness-induced bone loss in part through Rho signaling pathway.

Impact of Endothelial Cells and Mechanical Conditioning on Smooth Muscle Cell Extracellular Matrix Production and Differentiation

The aim of the current study was to explore the separate and coupled effects of endothelial cell (EC) presence and mechanical conditioning on smooth muscle cell (SMC) responses by combining bilayered poly(ethylene glycol) diacrylate (PEGDA) hydrogels with a pulsatile flow bioreactor. Each construct was composed of an outer PEGDA layer containing SMC and an inner PEGDA layer, either with or without EC. After an initial 3 days of static culture, EC(+) and EC(-) constructs were each further divided into two subgroups, half of which received mechanical conditioning mimetic of late gestation (mean pressures of approximately 50 mmHg and peak-to-trough pressure differentials of approximately 20 mmHg at approximately 140-180 beats/min) and half of which were cultured statically. After 18 additional days of culture, the SMC-containing layer of each construct was harvested, and western blots and quantitative histology were conducted to compare collagen type I, collagen type III, and elastin levels among treatment groups. SMC differentiation was evaluated by focusing on SMC marker calponin h1 and direct regulators of its gene expression-the transcription factor serum response factor (SRF) and two of its binding partners, myocardin and Elk-1. Combined EC and pulsatile flow conditioning increased elastin production, but decreased collagen type I deposition. Further, combined EC presence and mechanical stimulation increased SRF levels and the ratio of myocardin to active, phosphorylated Elk-1. This modulation of SRF and its binding partners appeared to result in a net increase in SMC differentiation, as evidenced by an associated increase in calponin h1 levels.

A new approach for selective rat endolymphatic sac epithelium collection to obtain pure specific RNA

The endolymphatic sac (ES) is an organ that is located in the temporal bone. Its anatomical location makes ES tissue collection without any contamination very difficult, and sometimes accurate molecular analyses of the ES are prevented due to this matter. In the present study, a new selective ES epithelial tissue collection method was attempted using laser capture microdissection to obtain pure ES RNA without any contamination. The validity of this method was demonstrated by RT-PCR with three specific primer pairs against osteocalcin, calponin H1, and NKCC2, which are specific proteins in bone, smooth muscle, and kidney/ES cells, respectively. From the RT-PCR results, the high specificity and sufficient sensitivity of the new method was indicated. It is considered that the new method is optimal for ES collection without contamination and it will be able to contribute to future analyses of the ES.

Role of H2-calponin in Regulating Macrophage Motility and Phagocytosis

The actin cytoskeleton plays a major role in cell motility that is essential for the function of phagocytes. Calponin is an actin-associated regulatory protein. Here we report the finding of significant levels of the h2 isoform of calponin in peripheral blood cells of myeloid lineage. To study the functional significance, h2-calponin gene (Cnn2) interrupted mice were constructed. Germ line transmission of the Cnn2-flox-neo allele was obtained in chimeras from two independent clones of targeted embryonic stem cells. The insertion of the neo(R) cassette into intron 2 of the Cnn2 gene resulted in a significant knockdown of h2-calponin expression. Removing the frt-flanked neo(R) cassette by FLP1 recombinase rescued the knockdown effect. Cre recombinase-induced deletion of the loxP-flanked exon 2 eliminated the expression of h2-calponin protein. H2-calponin-free mice showed reduced numbers of peripheral blood neutrophils and monocytes. H2-calponin-free macrophages demonstrated a higher rate of proliferation and faster migration than that of h2-calponin-positive cells, consistent with a faster diapedesis of peripheral monocytes and neutrophils. H2-calponin-free macrophages showed reduced spreading in adhesion culture together with decreased tropomyosin in the actin cytoskeleton. The lack of h2-calponin also significantly increased macrophage phagocytotic activity, suggesting a novel mechanism to regulate phagocyte functions.

Reduction of Calponin h1 expression in human colon cancer blood vessels

Aims Calponin h1 (CN) is a differentiation marker of smooth muscle cells that has been reported to be down-regulated in the blood vessels of several human tumors. In this study, we examined CN expression in blood vessels in relation to the clinical and pathological features of colon cancer tissue samples. Methods Fifty-six patients who had undergone colectomy for colon cancer were examined. To assess patients' disease-free survival, those who had metastasis at the time of surgical operation were excluded. Immunohistochemistry was performed by the indirect immunoperoxidase method, using serial sections made from formalin fixed and paraffin embedded tissue blocks. Results We found that the expression of vascular CN in the peripheral region of colon cancer tissues was significantly reduced in association with tumor progression, lymphatic invasion, vascular invasion and recurrence. This reduction of CN indicated not only a decrease of pericytes and/or smooth muscle cells in tumor vessels, but also the immaturity of those cells, since CN down-regulation occurred even in α-smooth muscle actin-positive cells. The down-regulation of CN in vessels in the peripheral region of tumor tissues was inversely associated with the expression of VEGF (vascular endothelial growth factor), seemingly advantageous to angiogenesis. Conclusion The down-regulation of CN expression in colon cancer vasculature evaluated by immunohistochemistry may be useful in conjunction with conventional staging procedures to predict more reliable outcome and to select therapeutic treatment.