Anabolic Proteins Research Articles

Small Molecules Alone or in Combination to Treat Joint Disease and Progress Toward Gene Therapy

The field of biologics for joint disease, specifically articular cartilage injury and osteoarthritis, is one that has stimulated much excitement in the area of orthopaedics. The deficit of growth factors and anticatabolics in joint disease is now a well-recognized problem; however, unraveling the mystery associated with appropriate dose and timing of administration and when these molecules might have their most therapeutic benefits would be the subject of study for decades to come. Further, the technique of how these proteins are enhanced in the joint to result in the greatest influence remains in question. Shall we deliver individual therapeutic proteins in one dose? Shall we enhance the joint environment through biologic solutions that mimic the body's response to injury? Or shall we strive to maintain continued elevated levels of these proteins through gene therapeutic strategies given the timeline of cartilage healing is slow and can sometimes require months to years to heal? The scientific community recognizes that the short half-life of recombinant proteins combined with the question of whether one individual protein can influence healing to the point of augmenting cartilage repair or change the outcome of an osteoarthritic joint leads to asking bigger questions such as what can we attain in multiple combinations of these factors and what is the best way to enhance the joint environment. The evidence is mounting in various studies that increasing levels of anabolic and anticatabolic proteins may have influential therapeutic effects in joints and may change the outcome of cartilage injury and osteoarthritis. This is a summary of the common biologic approaches to enhancing various proteins that may have important effects in joint disease.

MTOR inhibition activates overall protein degradation by the ubiquitin proteasome system as well as by autophagy

Growth factors and nutrients enhance protein synthesis and suppress overall protein degradation by activating the protein kinase mammalian target of rapamycin (mTOR). Conversely, nutrient or serum deprivation inhibits mTOR and stimulates protein breakdown by inducing autophagy, which provides the starved cells with amino acids for protein synthesis and energy production. However, it is unclear whether proteolysis by the ubiquitin proteasome system (UPS), which catalyzes most protein degradation in mammalian cells, also increases when mTOR activity decreases. Here we show that inhibiting mTOR with rapamycin or Torin1 rapidly increases the degradation of long-lived cell proteins, but not short-lived ones, by stimulating proteolysis by proteasomes, in addition to autophagy. This enhanced proteasomal degradation required protein ubiquitination, and within 30 min after mTOR inhibition, the cellular content of K48-linked ubiquitinated proteins increased without any change in proteasome content or activity. This rapid increase in UPS-mediated proteolysis continued for many hours and resulted primarily from inhibition of mTORC1 (not mTORC2), but did not require new protein synthesis or key mTOR targets: S6Ks, 4E-BPs, or Ulks. These findings do not support the recent report that mTORC1 inhibition reduces proteolysis by suppressing proteasome expression [Zhang Y, et al. (2014) Nature 513(7518):440-443]. Several growth-related proteins were identified that were ubiquitinated and degraded more rapidly after mTOR inhibition, including HMG-CoA synthase, whose enhanced degradation probably limits cholesterol biosynthesis upon insulin deficiency. Thus, mTOR inhibition coordinately activates the UPS and autophagy, which provide essential amino acids and, together with the enhanced ubiquitination of anabolic proteins, help slow growth.

Resistance Training With Excessive Training Load and Insufficient Recovery Alters Skeletal Muscle Mass–Related Protein Expression

The aim of this study was to investigate the effects of a resistance training program with excessive training load and insufficient recovery time between bouts on muscle hypertrophy- and atrophy-related protein expression. Male Wistar rats were randomly assigned to either a trained (TR, N = 9) or a sedentary (SE, N = 9) group. The TR group was subjected to a 12-week resistance training program with excessive training load and insufficient recovery between bouts that was designed to induce plantaris muscle atrophy. After the 12-week experiment, the plantaris muscle was collected to analyze the cross-sectional area (CSA) of the muscle fibers, and MAFbx, MyoD, myogenin, and IGF-I protein expression (Western blot). The CSA was reduced significantly (-17%, p ≤ 0.05) in the TR group compared with the SE group. Reciprocally, there was a significant (p ≤ 0.05) 20% increase in MAFbx protein expression, whereas the MyoD (-27%), myogenin (-29%), and IGF-I (-43%) protein levels decreased significantly (p ≤ 0.05) in the TR group compared with the SE group. In conclusion, our data indicated that muscle atrophy induced by resistance training with excessive training load and insufficient recovery was associated with upregulation of the MAFbx catabolic protein and downregulation of the MyoD, myogenin, and IGF-I anabolic proteins. These findings suggest that quantitative analysis of these proteins can be important and complementary with other biochemical markers to confirm a possible overtraining diagnosis.

Functional Proteome of Plasma to Distinguish Anabolic Responsive Proteins in an Estrogen Deficiency Model of Osteoporosis by Treatment With an Anabolic Agent PTH

Functional Proteome of Plasma to Distinguish Anabolic Responsive Proteins in an Estrogen Deficiency Model of Osteoporosis by Treatment With an Anabolic Agent PTH

Endogenous Bioactive Proteins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Intervertebral Discs: Identification of Potential Target Proteins for Gene Therapeutic Approaches

Introduction Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic, and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. Materials and Methods NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56/range 29-84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for 4 weeks. Subsequently, cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Results Independent from gender, age, and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β, and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6, and 7; insulin-like growth factor 1; and transforming growth factor β1 and 3, however, remained below the minimal detectable quantities. Conclusion The advantage of the relative mass value evaluation method is the ability to perform precise quantifications of protein concentrations. To our knowledge, this method has not been applied to analyze a broad array of endogenously expressed bioactive factors in NP cells from a larger number of human specimens. The findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases. Disclosure of Interest None declared

Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene.

Fluid shear stress and melatonin in combination activate anabolic proteins in MC3T3‐E1 osteoblast cells

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal-regulated kinase (p-ERK) in MC3T3-E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1 hr) and melatonin (1 mM) in combination reduced autophagic marker LC3-II compared with fluid shear stress (1 hr) and/or melatonin (0.1 mM). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p-ERK, phosphorylation of serine-threonine protein kinase (p-Akt), phosphorylation of mammalian target of rapamycin (p-mTOR), and p85-S6K were investigated. p-Akt, p-mTOR (Ser 2481) expressions increased with the addition of 1 mM melatonin prior to 0.1 mM melatonin treatment. However, p-S6K expression did not change significantly. Next, mitochondria activity including Bcl-2, Bax, catalase, and Mn-superoxide dismutase (Mn-SOD) were studied. Expressions of Bcl-2, Bax, and catalase proteins were low under fluid shear stress plus 1 mM melatonin compared with only fluid shear stress alone, whereas Mn-SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4 hr) and melatonin (0.1, 1 mM). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p-ERK in MC3T3-E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts.

Review of the Currently Available Systems to Obtain Platelet Related Products to Treat Equine Musculoskeletal Injuries

Lameness secondary to chronic or acute injuries of the musculoskeletal system is a cause of equine wastage. This old problem has been traditionally managed with treatments, such as corticosteroids and non steroidal antiinflammatory drugs. However, recent discoveries in the pathophysiology of these diseases have provided new therapeutic approaches to their treatments. The stimulation of cellular proliferation with increase in the anabolic capacity of the resident cells for producing a good quality extracellular matrix represents a real avenue for the treatment of these diseases in horses. The injection of platelet related products has won great acceptation between equine practitioners, since these substances can be obtained from the same patient by using different procedures or devices in field conditions. These substances are source of anti-inflammatory, angiogenic and anabolic proteins (i.e., growth factors and cytokines) and cells with immunomodulatory activity (i.e., lymphocytes and macrophages). However, the cellular and molecular content of these substances depend on the method or kit used for their preparation. A review about patented and non patented technologies and procedures for the treatment of musculoskeletal diseases in horses by using platelet related products is presented in this article. Keywords: Autologous platelet concentrates, chronic musculoskeletal diseases, desmopathy, devices, growth factors, horse, osteoarthritis, platelet gel, platelet rich plasma, tendinopathy

A step towards underpinning the molecular signalling events regulating muscle protein loss in critically ill patients

A step towards underpinning the molecular signalling events regulating muscle protein loss in critically ill patients

Regulation of gene expression in intervertebral disc cells by low and high hydrostatic pressure

Intervertebral disc structures are exposed to wide ranges of intradiscal hydrostatic pressure during different loading exercises and are at their minimum during lying or relaxed sitting and at maximum during lifting weights with a round back. We hypothesize that these different loading magnitudes influence the intervertebral disc (IVD) by alteration of disc matrix turnover depending on their magnitudes. Therefore the aim of this study was to assess changes in gene expression of human nucleus cells after the application of low hydrostatic pressure (0.25 MPa) and high hydrostatic pressure (2.5 MPa). IVD cells isolated from the nucleus of human (n = 18) and bovine (n = 24 from four animals) disc biopsies were seeded into three-dimensional collagen type-I matrices and exposed to the different loading magnitudes by specially developed pressure chambers. The lower pressure range (0.25 MPa, 30 min, 0.1 Hz) was applied with a recently published device by using an external compression cylinder. For the application of higher loads (2.5 MPa, 30 min, 0.1 Hz) the cell-loaded collagen gels were sealed into sterile bags with culture medium and stimulated in a newly developed water-filled compression cylinder by using a loading frame. These methods allowed the comparison of loading regimes in a wide physiological range under an equal three-dimensional culture conditions. Cells were harvested 24 h after the end of stimulation and changes in the expression of genes known to influence IVD matrix turnover (collagen-I, collagen-II, aggrecan, MMP1, MMP2, MMP3, MMP13) were analyzed by real-time RT-PCR. A Wilcoxon signed-rank test(1) and a Wilcoxon 2-sample test(2) were performed to detect differences between the stimulated and control samples(1) and differences between low and high hydrostatic pressure(2). Multiple testing was considered by adjusting the p value appropriately. Both regimes of hydrostatic pressure influenced gene expression in nucleus cells with opposite tendencies for the matrix forming proteins aggrecan and collagen type-I in response to the two different pressure magnitudes: Low hydrostatic-pressure (0.25 MPa) tended to increase collagen-I and aggrecan expression of human nucleus cells (P < 0.05) but only to a small degree. High hydrostatic pressure (2.5 MPa) tended to decrease gene expression of all anabolic proteins with significant effects on aggrecan expression of nucleus cells (P = 0.004). Low hydrostatic pressure had no influence on the expression of matrix metalloproteinases (MMP1, MMP2, MMP3 and MMP13). In contrast, high hydrostatic pressure tended to increase the expression of MMP1, MMP3 and MMP13 of human nucleus cells with high individual-individual variations. The decreased expression of aggrecan (P = 0.008) and collagen type II (P = 0.023) and the increased MMP3 expression (P = 0.008) in response to high hydrostatic pressure could be confirmed in additional experiments with bovine nucleus cells. These results suggest that hydrostatic pressure as one of the physiological stimuli of the IVD may influence matrix turnover in a magnitude dependent way. Low hydrostatic pressure (0.25 MPa) has quite small influences with a tendency to anabolic effects, whereas high hydrostatic pressure (2.5 MPa) tends to decrease the matrix protein expression with a tendency to increase some matrix-turnover enzymes. Therefore, hydrostatic pressure may regulate disc matrix turnover in a dose-dependent way.

Longitudinal changes of insulin-like growth factors and their binding proteins throughout normal pregnancy.

Insulin-like growth factors (IGFs) are anabolic proteins that are essential regulators of cell division, differentiation and growth. We describe the longitudinal changes in IGF-I, IGF-II and the binding proteins IGFBP-1, -2 and -3 before and during normal pregnancy. Serum samples were taken before conception and then at 12, 24 and 36 weeks of gestation in 41 healthy women with uncomplicated pregnancies. We measured IGF-I using an automated chemiluminescent method, IGF-II and IGFBP-2 using in-house radioimmunoassays (RIAs), and IGFBP-1 and IGFBP-3 using commercial enzyme-linked immunosorbent assay (ELISA) and RIA kits, respectively. Because of the potential haemodilution effects during pregnancy, albumin was also measured in all samples. There was a significant fall in IGF-I during the first (36%) and second trimesters (21%) followed by an increase of 25% at 36 weeks. During pregnancy, the mean IGF-II concentrations fell by 12% at 12 weeks, 8% at 24 and 8% at 36 weeks compared with pre-conception values. When IGF-II results were adjusted for the haemodilution of pregnancy, its concentrations increased. During pregnancy, there was a rapid increase in mean IGFBP-1 levels by 17-fold (12 weeks), 24-fold (24 weeks) and 25-fold (36 weeks). IGFBP-2 concentrations fell after conception but started to increase towards term. This increase was more significant when adjusted for haemodilution. In contrast, IGFBP-3 concentrations increased significantly throughout pregnancy. Our data on the physiological changes of IGFs and their binding proteins add further evidence of the vital roles of these hormones throughout normal pregnancy.

Mammary Hypertrophy Is Not Associated with Increased Estrogen Receptors

Estrogen promotes secondary female sex characteristics, including breast enlargement. Since excessive breast hypertrophy is unrelated to elevated serum estrogen levels, it has been postulated that the enlarged breast is a hypersensitive "target organ." At the cellular level, estrogen crosses the cell membrane, is bound to a cytoplasmic estrogen receptor (ER), and induces the formation of specific anabolic proteins. In breast cancer, this estrogen receptor is regarded as a measure of the sensitivity of the cell to estrogen. To determine if mammary hypertrophy is related to an increase in the number of estrogen receptors, we assayed breast tissue, not fat, from 25 consecutive breast reductions. The median age of patients was 26 years (17 to 77 years), with 752 gm per breast removed on average. Twenty-four percent of the patients were taking estrogens, primarily birth control bills. Cellular estrogen-receptor status was measured by a standardized cytosol extraction radioactive estradiol technique. Estrogen receptors were undetectable (less than 3 fmol/mg cytosol protein) in all patients. We conclude that estrogen receptors alone, and hence estrogen, are not a determinant in mammary hypertrophy. If the enlarged breast is a "target organ," it is by another mechanism.

Tumor necrosis factor-alpha inhibits growth hormone secretion from cultured anterior pituitary cells.

Anabolic proteins such as pituitary GH enhance the function of several immune cell types. The converse could also exist, that is communication from the immune cells to GH-producing somatotrophs. To test this hypothesis, tumor necrosis factor-alpha (TNF-alpha), a product of activated macrophages, was exposed to cultured rat pituitary cells, and GH release was monitored. TNF alpha inhibited basal and GH-releasing hormone-stimulated GH accumulation, with IC50 values of 170 U/ml (5.2 ng/ml) and 50 U/ml (1.5 ng/ml), respectively. This inhibition was first measured after 6 h of TNF alpha treatment, continued for at least 3 days, and was reversible. A number of measurements (e.g. trypan blue exclusion, chromium release, and GH cell content) yielded no signs of cytotoxicity to explain the inhibition. We conclude that TNF alpha can reduce basal and stimulated pituitary GH release in vitro.