Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells.

Mechano-growth factor peptide, the COOH terminus of unprocessed insulin-like growth factor 1, has no apparent effect on myoblasts or primary muscle stem cells.

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

Junctophilin 1 and 2 proteins interact with the L-type Ca2+ channel dihydropyridine receptors (DHPRs) in skeletal muscle.

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent studies have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle, JP1 and JP2 are part of a complex that, in addition to ryanodine receptor 1 (RyR1), includes caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knocked down for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type Ca2+ current accompanied by a reduced number of DHPRs at the plasma membrane, whereas there was no substantial alteration in Ca2+ release from the sterol regulatory element-binding protein. Altogether, these results suggest that JP1 and JP2 can facilitate the assembly of DHPR with other proteins of the excitation-contraction coupling machinery.

Angiopoietin-like 3-derivative LNA043 for cartilage regeneration in osteoarthritis: a randomized phase 1 trial.

Osteoarthritis (OA) is a common, debilitating, chronic disease with no disease-modifying drug approved to date. We discovered LNA043-a derivative of angiopoietin-like 3 (ANGPTL3)-as a potent chondrogenesis inducer using a phenotypic screen with human mesenchymal stem cells. We show that LNA043 promotes chondrogenesis and cartilage matrix synthesis in vitro and regenerates hyaline articular cartilage in preclinical OA and cartilage injury models in vivo. LNA043 exerts at least part of these effects through binding to the fibronectin receptor, integrin α5ß1 on mesenchymal stem cells and chondrocytes. In a first-in-human (phase 1), randomized, double-blinded, placebo-controlled, single ascending dose, single-center trial ( NCT02491281 ; sponsored by Novartis Pharmaceuticals), 28 patients with knee OA were injected intra-articularly with LNA043 or placebo (3:1 ratio) either 2 h, 7 d or 21 d before total knee replacement. LNA043 met its primary safety endpoint and showed short serum pharmacokinetics, cartilage penetration and a lack of immunogenicity (secondary endpoints). Post-hoc transcriptomics profiling of cartilage revealed that a single LNA043 injection reverses the OA transcriptome signature over at least 21 d, inducing the expression of hyaline cartilage matrix components and anabolic signaling pathways, while suppressing mediators of OA progression. LNA043 is a novel disease-modifying OA drug candidate that is currently in a phase 2b trial ( NCT04864392 ) in patients with knee OA.

SHP-2 activates signaling of the nuclear factor of activated T cells to promote skeletal muscle growth.

The formation of multinucleated myofibers is essential for the growth of skeletal muscle. The nuclear factor of activated T cells (NFAT) promotes skeletal muscle growth. How NFAT responds to changes in extracellular cues to regulate skeletal muscle growth remains to be fully defined. In this study, we demonstrate that mice containing a skeletal muscle-specific deletion of the tyrosine phosphatase SHP-2 (muscle creatine kinase [MCK]-SHP-2 null) exhibited a reduction in both myofiber size and type I slow myofiber number. We found that interleukin-4, an NFAT-regulated cytokine known to stimulate myofiber growth, was reduced in its expression in skeletal muscles of MCK-SHP-2-null mice. When SHP-2 was deleted during the differentiation of primary myoblasts, NFAT transcriptional activity and myotube multinucleation were impaired. Finally, SHP-2 coupled myotube multinucleation to an integrin-dependent pathway and activated NFAT by stimulating c-Src. Thus, SHP-2 transduces extracellular matrix stimuli to intracellular signaling pathways to promote skeletal muscle growth.

STING regulates peripheral nerve regeneration and colony stimulating factor 1 receptor (CSF1R) processing in microglia.

Inflammatory responses are crucial for regeneration following peripheral nerve injury (PNI). PNI triggers inflammatory responses at the site of injury. The DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream effector stimulator of interferon genes (STING) sense foreign and self-DNA and trigger type I interferon (IFN) immune responses. We demonstrate here that following PNI, the cGAS/STING pathway is upregulated in the sciatic nerve of naive rats and dysregulated in old rats. In a nerve crush mouse model where STING is knocked out, myelin content in sciatic nerve is increased resulting in accelerated functional axon recovery. STING KO mice have lower macrophage number in sciatic nerve and decreased microglia activation in spinal cord 1 week post injury. STING activation regulated processing of colony stimulating factor 1 receptor (CSF1R) and microglia survival in vitro. Taking together, these data highlight a previously unrecognized role of STING in the regulation of nerve regeneration.

A High-Throughput Screening Identifies MICU1 Targeting Compounds.

Mitochondrial Ca2+ uptake depends on the mitochondrial calcium uniporter (MCU) complex, a highly selective channel of the inner mitochondrial membrane (IMM). Here, we screen a library of 44,000 non-proprietary compounds for their ability to modulate mitochondrial Ca2+ uptake. Two of them, named MCU-i4 and MCU-i11, are confirmed to reliably decrease mitochondrial Ca2+ influx. Docking simulations reveal that these molecules directly bind a specific cleft in MICU1, a key element of the MCU complex that controls channel gating. Accordingly, in MICU1-silenced or deleted cells, the inhibitory effect of the two compounds is lost. Moreover, MCU-i4 and MCU-i11 fail to inhibit mitochondrial Ca2+ uptake in cells expressing a MICU1 mutated in the critical amino acids that forge the predicted binding cleft. Finally, these compounds are tested ex vivo, revealing a primary role for mitochondrial Ca2+ uptake in muscle growth. Overall, MCU-i4 and MCU-i11 represent leading molecules for the development of MICU1-targeting drugs.

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins.

We show here that beta1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The beta1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, beta1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, beta1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the beta1 cytodomain plays an important role in mediating beta1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, beta1A associates with IRS-1 and beta1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.

Tough Composite Hydrogels with High Loading and Local Release of Biological Drugs.

Hydrogels are under active development for controlled drug delivery, but their clinical translation is limited by low drug loading capacity, deficiencies in mechanical toughness and storage stability, and poor control over the drug release that often results in burst release and short release duration. This work reports a design of composite clay hydrogels, which simultaneously achieve a spectrum of mechanical, storage, and drug loading/releasing properties to address the critical needs from translational perspectives. The clay nanoparticles provide large surface areas to adsorb biological drugs, and assemble into microparticles that are physically trapped within and toughen hydrogel networks. The composite hydrogels demonstrate feasibility of storage, and extended release of large quantities of an insulin-like growth factor-1 mimetic protein (8 mg mL-1 ) over four weeks. The release rate is primarily governed by ionic exchange and can be upregulated by low pH, which is typical for injured tissues. A rodent model of Achilles tendon injury is used to demonstrate that the composite hydrogels allow for highly extended and localized release of biological drugs in vivo, while demonstrating biodegradation and biocompatibility. These attributes make the composite hydrogel a promising system for drug delivery and regenerative medicine.

Safety, tolerability, and preliminary efficacy of an IGF-1 mimetic in patients with spinal and bulbar muscular atrophy: a randomised, placebo-controlled trial.

BACKGROUND: Spinal and bulbar muscular atrophy is an X-linked neuromuscular disease caused by CAG repeat expansion in the androgen receptor gene. Patients with this disease have low concentrations of insulin-like growth factor-1 (IGF-1), and studies of overexpression and administration of IGF-1 showed benefit in a transgenic model; thus the IGF-1 pathway presents as a potential treatment target. We assessed safety, tolerability, and preliminary efficacy of BVS857, an IGF-1 mimetic, in patients with spinal and bulbar muscular atrophy. METHODS: In this randomised, double-blind, placebo-controlled trial, we recruited patients from neuromuscular centres in Denmark (Copenhagen), Germany (Ulm), Italy (Padova), and three sites within the USA (Bethesda, MD; Irvine, CA; and Columbus, OH). Eligible patients were 18 years or older with a confirmed genetic diagnosis of spinal and bulbar muscular atrophy, were ambulatory, had symptomatic weakness, and had serum IGF-1 concentrations of 170 ng/mL or lower. Patients were randomly assigned (2:1) to study drug or placebo by a number scheme. Patients, investigators, and study personnel were masked to treatment assignment. After a safety and tolerability assessment with eight patients, BVS857 was administered once a week (0·06 mg/kg intravenously) for 12 weeks. Primary outcome measures were safety, tolerability, and the effects of BVS857 on thigh muscle volume (TMV) measured by MRI. The ratio of TMV at day 85 to baseline was analysed with ANCOVA per protocol. Secondary outcomes of muscle strength and function were measured with the Adult Myopathy Assessment Tool, lean body mass through dual energy x-ray absorptiometry, and BVS857 pharmacokinetics. This trial was registered with ClinicalTrials.gov, NCT02024932. FINDINGS: 31 patients were assessed for eligibility, 27 of whom were randomly assigned to either BVS857 treatment (n=18) or placebo (n=9), and 24 were included in the preliminary efficacy analysis (BVS857 group, n=15; placebo group, n=9). BVS857 was generally safe with no serious adverse events. No significant differences were found in adverse events between the BVS857 and placebo groups. Immunogenicity was detected in 13 (72%) of 18 patients in the BVS857 group, including crossreacting antibodies with neutralising capacity to endogenous IGF-1 in five patients. TMV decreased from baseline to day 85 in the placebo group (-3·4% [-110 cm3]) but not in the BVS857 group (0% [2 cm3]). A significant difference in change in TMV was observed in the BVS857 group versus the placebo group (geometric-mean ratio 1·04 [90% CI 1·01-1·07]; p=0·02). There were no differences between groups in measures of muscle strength and function. INTERPRETATION: TMV remained stable in patients with spinal and bulbar muscular atrophy after being given BVS857 for 12 weeks. The intervention was associated with high incidence of immunogenicity and did not improve muscle strength or function. Additional studies might be needed to assess the efficacy of activating the IGF-1 pathway in this disease. FUNDING: Novartis Pharmaceuticals and the US National Institutes of Health.

SHP-2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway.

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.

ATP Citrate Lyase Regulates Myofiber Differentiation and Increases Regeneration by Altering Histone Acetylation.

ATP citrate lyase (ACL) plays a key role in regulating mitochondrial function, as well as glucose and lipid metabolism in skeletal muscle. We report here that ACL silencing impairs myoblast and satellite cell (SC) differentiation, and it is accompanied by a decrease in fast myosin heavy chain isoforms and MYOD. Conversely, overexpression of ACL enhances MYOD levels and promotes myogenesis. Myogenesis is dependent on transcriptional but also other mechanisms. We show that ACL regulates the net amount of acetyl groups available, leading to alterations in acetylation of H3(K9/14) and H3(K27) at the MYOD locus, thus increasing MYOD expression. ACL overexpression in murine skeletal muscle leads to improved regeneration after cardiotoxin-mediated damage. Thus, our findings suggest a mechanism for regulating SC differentiation and enhancing regeneration, which might be exploited for devising therapeutic approaches for treating skeletal muscle disease.

ATP citrate lyase improves mitochondrial function in skeletal muscle.

Mitochondrial dysfunction is associated with skeletal muscle pathology, including cachexia, sarcopenia, and the muscular dystrophies. ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes mitochondria-derived citrate into oxaloacetate and acetyl-CoA. Here we report that activation of ACL in skeletal muscle results in improved mitochondrial function. IGF1 induces activation of ACL in an AKT-dependent fashion. This results in an increase in cardiolipin, thus increasing critical mitochondrial complexes and supercomplex activity, and a resultant increase in oxygen consumption and cellular ATP levels. Conversely, knockdown of ACL in myotubes not only reduces mitochondrial complex I, IV, and V activity but also blocks IGF1-induced increases in oxygen consumption. In vivo, ACL activity is associated with increased ATP. Activation of this IGF1/ACL/cardiolipin pathway combines anabolic signaling with induction of mechanisms needed to provide required ATP.

Gαi2 signaling is required for skeletal muscle growth, regeneration, and satellite cell proliferation and differentiation.

We have previously shown that activation of Gαi2, an α subunit of the heterotrimeric G protein complex, induces skeletal muscle hypertrophy and myoblast differentiation. To determine whether Gαi2 is required for skeletal muscle growth or regeneration, Gαi2-null mice were analyzed. Gαi2 knockout mice display decreased lean body mass, reduced muscle size, and impaired skeletal muscle regeneration after cardiotoxin-induced injury. Short hairpin RNA (shRNA)-mediated knockdown of Gαi2 in satellite cells (SCs) leads to defective satellite cell proliferation, fusion, and differentiation ex vivo. The impaired differentiation is consistent with the observation that the myogenic regulatory factors MyoD and Myf5 are downregulated upon knockdown of Gαi2. Interestingly, the expression of microRNA 1 (miR-1), miR-27b, and miR-206, three microRNAs that have been shown to regulate SC proliferation and differentiation, is increased by a constitutively active mutant of Gαi2 [Gαi2(Q205L)] and counterregulated by Gαi2 knockdown. As for the mechanism, this study demonstrates that Gαi2(Q205L) regulates satellite cell differentiation into myotubes in a protein kinase C (PKC)- and histone deacetylase (HDAC)-dependent manner.

Gαi2 signaling promotes skeletal muscle hypertrophy, myoblast differentiation, and muscle regeneration.

Skeletal muscle atrophy results in loss of strength and an increased risk of mortality. We found that lysophosphatidic acid, which activates a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor, stimulated skeletal muscle hypertrophy through activation of Gα(i2). Expression of a constitutively active mutant of Gα(i2) stimulated myotube growth and differentiation, effects that required the transcription factor NFAT (nuclear factor of activated T cells) and protein kinase C. In addition, expression of the constitutively active Gα(i2) mutant inhibited atrophy caused by the cachectic cytokine TNFα (tumor necrosis factor-α) by blocking an increase in the abundance of the mRNA encoding the E3 ubiquitin ligase MuRF1 (muscle ring finger 1). Gα(i2) activation also enhanced muscle regeneration and caused a switch to oxidative fibers. Our study thus identifies a pathway that promotes skeletal muscle hypertrophy and differentiation and demonstrates that Gα(i2)-induced signaling can act as a counterbalance to MuRF1-mediated atrophy, indicating that receptors that act through Gα(i2) might represent potential targets for preventing skeletal muscle wasting.

SHP-2 regulates the phosphatidylinositide 3'-kinase/Akt pathway and suppresses caspase 3-mediated apoptosis.

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. The ability of SHP-2 to regulate the PI3K/Akt pathway is suggested to result in the positive effect of SHP-2 on cell survival. Whether SHP-2 regulates insulin-like growth factor-1 (IGF-1)-dependent activation of Akt at the level of PI3K has yet to be established. Furthermore, the identification of the down-stream apoptotic target engaged by SHP-2 in cell survival also has yet to be determined. Here, we show that overexpression of a catalytically inactive mutant of SHP-2 inhibited insulin-like growth factor-1 (IGF-1)-dependent PI3K and Akt activation. Consistent with the observation that SHP-2 participates in pro-survival signaling fibroblasts expressing a deletion within exon 3 of SHP-2, which results in a truncation of the amino-terminus SH2 domain (SHP-2(Ex3-/-)), were hypersensitive to etoposide-induced cell death. SHP-2(Ex3-/-) fibroblasts exhibited enhanced levels of etoposide-induced caspase 3 activity as compared to wild-type fibroblasts and the enhanced level of caspase 3 activity was suppressed by a caspase 3-specific inhibitor. Re-introduction of wild-type SHP-2 into the SHP-2(Ex3-/-) fibroblasts rescued the hypersensitivity to etoposide-induced caspase 3 activation. The effects of abrogating SHP-2 function on cell survival were not specific to the loss of the amino-terminus SH2 domain of SHP-2 since RNAi-mediated knock-down of SHP-2 also reduced cell survival. Taken together, these data indicate that the catalytic activity of SHP-2 is required to regulate the PI3K/Akt pathway and thus likely participates in anti-apoptotic signaling by suppressing caspase 3-mediated apoptosis.

Fibronectin protects prostate cancer cells from tumor necrosis factor-alpha-induced apoptosis via the AKT/survivin pathway.

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84--> Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via beta1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-alpha-induced apoptosis. Evidence is provided that expression of beta1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-alpha-induced apoptosis. In contrast, the beta1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-alpha in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis.