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Cell Structure and Function Feb 1993We have previously shown that mouse L.P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we...
Characterization of cell-adhesive proteins secreted by cell lines growing in protein- and lipid-free synthetic medium: mouse L.P3 cells secrete a procollagen molecule with potent cell-attachment activity.
We have previously shown that mouse L.P3 cells secrete fibronectin and a novel protein, gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). Here, we screened and characterized cell-adhesive proteins in the conditioned media (CM's) of a series of cell lines growing in a protein- and lipid-free synthetic medium (P3 cell lines). Although cell-attachment activity of the CM's ranged from undetectable to highly significant, fractionation with affinity columns revealed the presence of significant cell-attachment activity in all CM's. Using cell-attachment assay and immunoassay on blotted filters, various cell-adhesive proteins were detected in the CM's, and most of them were identified as fibronectin, laminin, vitronectin, and collagen. GNCP-like proteins were detected in the CM's of HeLa.P3, JTC-16.P3, L.P3, and JTC-12.P3. There was no relationship between the origin of the cell lines and the cell-adhesive proteins secreted. GNCP purified from L.P3-CM was separated into 120-, 140-, 150-, and 160-kDa proteins on SDS-PAGE, which were judged to be a type of mouse type I procollagen from the following results: (1) they were digested by collagenase, (2) pepsin treatment converted the 150- and 160-kDa proteins into 120- and 140-kDa proteins, (3) they were recognized by anti-type I collagen antiserum, and (4) amino-terminal sequence of the pepsin-digested 140-kDa protein had significant homology with type I collagen. GNCP showed half-maximum activity of cell attachment at 0.03 micrograms/ml, indicating that GNCP was a cell-adhesive protein with an extremely high specific activity compared to other known cell-adhesive proteins.
Topics: Amino Acid Sequence; Animals; Blotting, Western; Cell Adhesion; Cell Adhesion Molecules; Cell Fractionation; Cell Line; Chromatography, Affinity; Collagenases; Culture Media, Conditioned; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibronectins; Heparin; Mice; Molecular Sequence Data; Molecular Weight; Pepsin A; Procollagen; Proteins
PubMed: 8504460
DOI: 10.1247/csf.18.61 -
Clinical Orthopaedics and Related... Apr 2023Joint contractures occur frequently after trauma or immobilization, but few reliable treatments are available. Extracorporeal shock wave therapy (ESWT) is often used for...
BACKGROUND
Joint contractures occur frequently after trauma or immobilization, but few reliable treatments are available. Extracorporeal shock wave therapy (ESWT) is often used for various musculoskeletal conditions, but whether it is effective for treating joint contractures and the mechanisms through which it might work for that condition remain unclear.
QUESTIONS/PURPOSES
Using a rat model, we asked, does ESWT (1) inhibit the progression of knee contracture, (2) ameliorate histopathologic joint changes, and (3) improve serum and myofascial fibrosis-related factors? We also asked, (4) what is the possible mechanism by which ESWT inhibits knee contracture?
METHODS
Thirty-two male Sprague-Dawley rats (12 weeks old and weighing 300 to 400 g) were randomly separated into two groups: control group (eight rats) and noncontrol group (24) in the first week. Rats in the control group were kept free in cages for 4 weeks, and the right lower limbs of the rats in the noncontrol group were immobilized in plaster for 4 weeks. ROM was then measured for each rat with or without 4 weeks of immobilization. After ROM measurement, rats in the noncontrol group were randomly separated into three groups: immobilization group (eight rats), remobilization group (eight rats), and remobilization with ESWT group (eight rats) at Week 4. Knee contracture was induced in rats by fixing the right knee with a plaster cast as in a previous study. The plaster cast was removed after 4 weeks; knee contracture was established when passive ROM was decreased and dysfunction such as abnormal gait occurred. Subsequently, rats with a remobilized joint contracture were treated with or without ESWT for 15 days (on Days 5, 10, and 15). The therapeutic effect was examined using ROM, joint diameter (as an indication of swelling), histopathologic changes, and the levels of fibrosis-related extracellular matrix component factors (hyaluronic acid, serum procollagen peptide, and laminin). The effect of ESWT on fibrosis protein was also evaluated using immunohistochemistry, quantitative polymerase chain reaction (qPCR), and Western blot. The expressions of factors in the TGF-β/SMADs pathway were also determined using Western blot and qPCR.
RESULTS
ESWT mitigated immobilization-induced knee contracture in rats by improving ROM (immobilization versus remobilization with ESWT: 53° ± 8° versus 32° ± 8° [95% confidence interval 13° to 30°]; p < 0.001) and joint swelling (immobilization versus remobilization with ESWT: 8 ± 0.8 cm versus 6 ± 0.3 cm [95% CI 0.4 to 2.2 cm]; p = 0.01). Histopathologic features of remission were alleviated after ESWT (immobilization versus remobilization with ESWT: thickness of the knee space: 0.2 ± 0.03 mm versus 0.6 ± 0.01 mm [95% CI -0.49 to -0.33 mm]; p < 0.001. On Masson staining, the positive expression area, which indicates collagen fiber deposition, was 24% ± 5% versus 9% ± 2% ([95% CI 10% to 21%]; p < 0.001). ESWT improved the serum fibrosis factors of hyaluronic acid, procollagen peptide, and laminin (immobilization versus remobilization with ESWT: hyaluronic acid: 412 ± 32 versus 326 ±15 ng/mL [95% CI 29 to 144 ng/mL]; p = 0.003; serum procollagen peptide: 19 ± 1 versus 12 ±1 ng/mL [95% CI 3 to 11 ng/mL]; p < 0.001; laminin: 624 ± 78 versus 468 ±9 ng/mL [95% CI 81 to 231 ng/mL]; p = 0.006) and myofascial factors of α-SMA and Type I collagen associated with immobilization-induced contractures.
CONCLUSION
The findings suggest that ESWT improved joint contracture by inhibiting the TGF-β1/SMADs signaling pathway in rats.
CLINICAL RELEVANCE
This work suggests ESWT may be worth exploring in preliminary research in humans to determine whether it may be a treatment option for patients with nontraumatic knee contractures. If the mechanism of ESWT can be confirmed in humans, ESWT might be a therapy for diseases involved in the TGF-β1/SMADs signaling pathway, such as hypertroic scarring and scleroderma.
Topics: Humans; Rats; Male; Animals; Transforming Growth Factor beta1; Extracorporeal Shockwave Therapy; Hyaluronic Acid; Laminin; Procollagen; Rats, Sprague-Dawley; Knee Joint; Contracture; Fibrosis; Range of Motion, Articular
PubMed: 36724201
DOI: 10.1097/CORR.0000000000002559 -
The Journal of Cell Biology Mar 1999Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage....
Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA procollagen fibrils labeled with antibodies to NH2-propeptide at approximately 70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB procollagen, and the newly synthesized type IIB collagen displaces the type IIA procollagen into the interterritorial matrix. To initiate studies on the function of type IIA procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-beta1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.
Topics: Base Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage; Cysteine; DNA Primers; Extracellular Matrix; Fluorescent Antibody Technique; Humans; Microscopy, Immunoelectron; Procollagen; Protein Binding; Protein Isoforms; Recombinant Proteins; Transforming Growth Factor beta
PubMed: 10085302
DOI: 10.1083/jcb.144.5.1069 -
Journal of Clinical Pharmacology Sep 2022We evaluated the pharmacokinetics, pharmacodynamics, and safety of a single subcutaneous dose of romosozumab 210 mg, a monoclonal antibody against sclerostin, in an...
We evaluated the pharmacokinetics, pharmacodynamics, and safety of a single subcutaneous dose of romosozumab 210 mg, a monoclonal antibody against sclerostin, in an open-label, parallel-group study in participants with severe (stage 4) renal impairment (RI; n = 8) or end-stage renal disease requiring hemodialysis (ESRD-RH; n = 8), or healthy participants with normal renal function (n = 8). Compared with the group with normal renal function, the mean romosozumab exposure was 31% and 43% higher as measured by maximum observed serum concentration and area under the concentration-time curve, respectively, in the severe RI group and similar to those in the ESRD-RH group. For all 3 groups, the maximum mean percent increase in procollagen type 1 N terminal propeptide and decrease in serum C-telopeptide levels from baseline were observed on day 15. Changes in procollagen type 1 N terminal propeptide and serum C-telopeptide were of similar patterns in all 3 groups. The single dose of romosozumab 210 mg was well tolerated. Adverse events (AEs) were reported for 13 patients (7 patients with severe RI and 6 with ESRD-RH), with no deaths, AEs, or serious AEs leading to withdrawal. The incidence of subjects with postbaseline transient decreases in serum calcium (severe RI, n = 1; ESRD-RH, n = 5) and increases in intact parathyroid hormone (severe RI, n = 7; ESRD-RH, n = 7; healthy, n = 3) were greater in severe RI and ESRD-RH groups than in the healthy group. All reported events of hypocalcemia (severe RI, n = 1; ESRD-RH, n = 4) were asymptomatic. These results support the use of romosozumab without dose adjustment in patients with severe RI or ESRD-RH.
Topics: Antibodies, Monoclonal; Area Under Curve; Humans; Kidney; Kidney Failure, Chronic; Procollagen; Renal Dialysis; Renal Insufficiency
PubMed: 35304747
DOI: 10.1002/jcph.2050 -
Clinical Chemistry and Laboratory... Feb 2016The aims of this study were to establish robust reference intervals and to investigate the factors influencing bone turnover markers (BTMs) in healthy premenopausal...
BACKGROUND
The aims of this study were to establish robust reference intervals and to investigate the factors influencing bone turnover markers (BTMs) in healthy premenopausal Spanish women.
METHODS
A total of 184 women (35-45 years) from 13 centers in Catalonia were analyzed. Blood and second void urine samples were collected between 8 a.m. and 10 a.m. after an overnight fast. Serum procollagen type I amino-terminal propeptide (PINP) and serum cross-linked C-terminal telopeptide of type I collagen (CTX-I) were measured by two automated assays (Roche and IDS), bone alkaline phosphatase (bone ALP) by ELISA, osteocalcin (OC) by IRMA and urinary NTX-I by ELISA. PTH and 25-hydroxyvitamin D (25OHD) levels were measured. All participants completed a questionnaire on lifestyle factors.
RESULTS
Reference intervals were: PINP: 22.7-63.1 and 21.8-65.5 μg/L, bone ALP: 6.0-13.6 μg/L, OC: 8.0-23.0 μg/L, CTX-I: 137-484 and 109-544 ng/L and NTX-I: 19.6-68.9 nM/mM. Oral contraceptive pills (OCPs) influenced PINP (p=0.007), and low body mass index (BMI) was associated with higher BTMs except for bone ALP. Women under 40 had higher median values of most BTMs. CTX-I was influenced by calcium intake (p=0.010) and PTH (p=0.007). 25OHD levels did not influence BTMs. Concordance between the two automated assays for PINP and particularly CTX-I was poor.
CONCLUSIONS
Robust reference intervals for BTMs in a Southern European country are provided. The effects of OCPs and BMI on their levels are significant, whilst serum 25OHD levels did not influence BTMs. Age, calcium intake, BMI and PTH influenced CTX-I. The two automated assays for measuring PINP and CTX-I are not interchangeable.
Topics: Adult; Alkaline Phosphatase; Biomarkers; Body Mass Index; Bone Remodeling; Collagen Type I; Enzyme-Linked Immunosorbent Assay; Female; Humans; Middle Aged; Osteocalcin; Parathyroid Hormone; Peptide Fragments; Peptides; Premenopause; Procollagen; Reference Values; Vitamin D
PubMed: 26088062
DOI: 10.1515/cclm-2015-0162 -
The Journal of Investigative Dermatology May 1988Sclerosing or morphea-like variant of basal cell carcinoma (BCC) is characterized by an extensive connective tissue stroma, and histopathology has suggested that the...
Selectively enhanced procollagen gene expression in sclerosing (morphea-like) basal cell carcinoma as reflected by elevated pro alpha 1(I) and pro alpha 1(III) procollagen messenger RNA steady-state levels.
Sclerosing or morphea-like variant of basal cell carcinoma (BCC) is characterized by an extensive connective tissue stroma, and histopathology has suggested that the extracellular matrix is largely composed of collagen. In addition, fibronectin deposition has been proposed to modulate tumor growth in BCC. In this study, we examined the expression of genes coding for type I, III, and IV procollagens, as well as for fibronectin, in tissue from 10 patients with sclerosing BCC. For comparison, tissues from 5 patients with nodular BCC and 4 controls were examined. Total RNA was isolated by CsCl density gradient centrifugation, and messenger RNA (mRNA) steady-state levels were determined by slot-blot hybridizations with human sequence specific complementary DNAs (cDNAs). The abundance of type I procollagen mRNA in sclerosing BCC tissue was increased to 233.6 +/- 36.7% of the controls (mean +/- SEM). The corresponding value for type III procollagen mRNA in sclerosing BCC was 281.8 +/- 54.8% of the controls. Consequently, the steady-state ratio of type I/III procollagen mRNAs in sclerosing BCCs (5.0 +/- 1.2; mean +/- SD) was within the control range. Thus, there is a coordinate increase in type I and type III procollagen mRNA levels in sclerosing BCC. In contrast, the values for type I and type III procollagen mRNAs in nodular BCC were not different from the controls. In addition, type IV procollagen and fibronectin mRNA levels were not different from the controls either in sclerosing or nodular BCCs, attesting to the selectivity of the increase in type I and III procollagen mRNA levels in sclerosing BCC. These observations may relate to the excessive deposition of the extracellular matrix stroma surrounding the tumor cells in sclerosing BCC.
Topics: Carcinoma, Basal Cell; Collagen; Fibronectins; Gene Expression Regulation; Homeostasis; Humans; Microscopy, Electron; Procollagen; RNA, Messenger; Reference Values; Scleroderma, Localized; Skin Neoplasms
PubMed: 3361139
DOI: 10.1111/1523-1747.ep12560782 -
The Journal of Biological Chemistry Nov 1988The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2...
Cleavage of type I and type II procollagens by type I/II procollagen N-proteinase. Correlation of kinetic constants with the predicted conformations of procollagen substrates.
The kinetic constants were examined for the cleavage of several types of procollagen by type I/II procollagen N-proteinase. The Km values were essentially the same (0.2 microM) for chick type I procollagen, human type I procollagen, and chick type II procollagen. However, the Vmax values differed over a 14-fold range. As reported previously, the enzyme did not cleave denatured type I or II procollagen. Also, it did not cleave human type III procollagen which contains the same scissle -Pro-Gln- bond as the pro-alpha 1(I) chain of type I procollagen. To explain the observations, Chou-Fasman rules were used to compare the secondary structures of the cleavage sites in the procollagens. The results supported a previous suggestion (Helseth, D. L., Jr., Lechner, J. L., and Veis, A. (1979) Biopolymers 18, 3005-3014) that the region carboxyl-terminal to cleavage site in the pro-alpha 1(I) chain of type I procollagen was in a hairpin conformation consisting of a beta-sheet, beta-turn, and beta-sheet. In both chick and human type I procollagen, the hairpin loop in the pro-alpha 1(I) chain consisted of about 18 amino acids. The cleavage site itself was in a short alpha-helical structure of four or five amino acids. The pro-alpha 2(I) chains had a similar hairpin loop of about 14 amino acids and alpha-helix of four or five amino acids containing the cleavage site. Chick type II procollagen, which had the highest Vmax value, had a longer hairpin structure of 22 amino acids, and the cleavage site was in a longer alpha-helical domain of 10 amino acids. In contrast, type III procollagen had a random-coil conformation in the same region. The results help to explain the unusual substrate requirements of type I/II N-proteinase. They also help explain why mutations that produce in-frame deletions of amino acids 84 or more residues carboxyl-terminal to the cleavage site make the protein resistant to the enzyme.
Topics: Amino Acid Sequence; Animals; Cattle; Chick Embryo; Endopeptidases; Humans; Kinetics; Molecular Sequence Data; Procollagen; Procollagen N-Endopeptidase; Protein Conformation; Rats; Time Factors
PubMed: 3053692
DOI: No ID Found -
ROS Scavenging and Anti-Wrinkle Effects of Clitocybin A Isolated from the Mycelium of the Mushroom .Journal of Microbiology and... May 2017Clitocybin A, an isoindolinone from , was investigated to assess its anti-wrinkle properties, through reactive oxygen species (ROS)-scavenging and elastase inhibitory...
Clitocybin A, an isoindolinone from , was investigated to assess its anti-wrinkle properties, through reactive oxygen species (ROS)-scavenging and elastase inhibitory activities, procollagen synthesis, and matrix metalloproteinase-1 (MMP-1) expression, in human primary dermal fibroblast-neonatal (HDF-N) cells. Clitocybin A exhibited no significant cytotoxicity up to 10 ppm in HDF-N cells, with cell viability and cell proliferation activity greater than 94.6% and 91.9%, respectively. Strong and concentration-dependent ROS radical scavenging activities of clitocybin A were observed following irradiation with UVB at 30 mJ/cm. Furthermore, clitocybin A treatment of cells at 0.1, 1, and 10 ppm exhibited decreased elastase activity, in a concentration-dependent manner, by 1.97%, 6.6%, and 8.31%, respectively, versus the control group. The effects of clitocybin A on procollagen synthesis and MMP-1 expression were investigated. Clitocybin A treatment of cells at 1, 5, and 10 ppm increased procollagen synthesis, by 67.9%, 74.4%, and 112.9%, respectively, versus the control group. At these concentrations, MMP-1 expression decreased significantly following UV irradiation. Together, these findings suggest that clitocybin A may be an effective ingredient for use in anti-wrinkle cosmetic products.
Topics: Agaricales; Cell Cycle; Cell Line; Cell Proliferation; Cell Survival; Cells, Cultured; Fibroblasts; Free Radical Scavengers; Humans; Isoindoles; Matrix Metalloproteinase 1; Mycelium; Pancreatic Elastase; Procollagen; Reactive Oxygen Species; Scattering, Radiation; Skin Aging; Ultraviolet Rays
PubMed: 28297750
DOI: 10.4014/jmb.1702.02050 -
Turk Kardiyoloji Dernegi Arsivi : Turk... Oct 2016
Topics: Cohort Studies; Electrocardiography; Humans; Hypertension; Peptide Fragments; Procollagen
PubMed: 27774961
DOI: 10.5543/tkda.2016.44450 -
The EMBO Journal Jan 2019Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in...
Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.
Topics: Animals; Autophagy; Calnexin; Cell Line; Endoplasmic Reticulum; Gene Knockdown Techniques; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Microtubule-Associated Proteins; Oryzias; Procollagen; Protein Folding
PubMed: 30559329
DOI: 10.15252/embj.201899847