Ciliary proteins specify the cell inflammatory response by tuning NFκB signalling, independently of primary cilia.

Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1ß, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation.This article has an associated First Person interview with the first author of the paper.

Heparan Sulfate Proteoglycan Synthesis Is Dysregulated in Human Osteoarthritic Cartilage.

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage loss and subchondral bone remodeling in response to abnormal mechanical load. Heparan sulfate (HS) proteoglycans bind to many proteins that regulate cartilage homeostasis, including growth factors, morphogens, proteases, and their inhibitors, and modulate their localization, retention, and biological activity. Changes in HS expression and structure may thus have important consequences for joint health. We analyzed normal and osteoarthritic human knee cartilage, and found HS biosynthesis was markedly disrupted in OA, with 45% of the 38 genes analyzed differentially regulated in diseased cartilage. The expression of several HS core proteins, biosynthesis, and modification enzymes was increased in OA cartilage, whereas the expression of the HS proteoglycans syndecan 4 and betaglycan was reduced. The structure of HS was also altered, with increased levels of 6-O-sulfation in osteoarthritic samples, which correlated with increased expression of HS6ST1, a 6-O-sulfotransferase, and GLCE, an epimerase that promotes 6-O-sulfation. siRNA silencing of HS6ST1 expression in primary OA chondrocytes inhibited extracellular signal-regulated kinase phosphorylation in response to fibroblast growth factor 2, showing that changes in 6-O-sulfation impact a key cartilage signaling pathway. Given the broad range of homeostatic and repair pathways that HS regulates, these changes in proteoglycan expression and HS structure are likely to have significant effects on joint health and progression of OA.

Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1-mediated endocytosis.

Matrix protease activity is fundamental to developmental tissue patterning and remains influential in adult homeostasis. In cartilage, the principal matrix proteoglycan is aggrecan, the protease-mediated catabolism of which defines arthritis; however, the pathophysiologic mechanisms that drive aberrant aggrecanolytic activity remain unclear. Human ciliopathies exhibit altered matrix, which has been proposed to be the result of dysregulated hedgehog signaling that is tuned within the primary cilium. Here, we report that disruption of intraflagellar transport protein 88 (IFT88), a core ciliary trafficking protein, increases chondrocyte aggrecanase activity in vitro. We find that the receptor for protease endocytosis in chondrocytes, LDL receptor-related protein 1 (LRP-1), is unevenly distributed over the cell membrane, often concentrated at the site of cilia assembly. Hypomorphic mutation of IFT88 disturbs this apparent hot spot for protease uptake, increases receptor shedding, and results in a reduced rate of protease clearance from the extracellular space. We propose that IFT88 and/or the cilium regulates the extracellular remodeling of matrix-independently of Hedgehog regulation-by enabling rapid LRP-1-mediated endocytosis of proteases, potentially by supporting the creation of a ciliary pocket. This result highlights new roles for the cilium's machinery in matrix turnover and LRP-1 function, with potential relevance in a range of diseases.-Coveney, C. R., Collins, I., Mc Fie, M., Chanalaris, A., Yamamoto, K., Wann, A. K. T. Cilia protein IFT88 regulates extracellular protease activity by optimizing LRP-1-mediated endocytosis.

Suramin Inhibits Osteoarthritic Cartilage Degradation by Increasing Extracellular Levels of Chondroprotective Tissue Inhibitor of Metalloproteinases 3.

Osteoarthritis is a common degenerative joint disease for which no disease-modifying drugs are currently available. Attempts to treat the disease with small molecule inhibitors of the metalloproteinases that degrade the cartilage matrix have been hampered by a lack of specificity. We aimed to inhibit cartilage degradation by augmenting levels of the endogenous metalloproteinase inhibitor, tissue inhibitor of metalloproteinases (TIMP)-3, through blocking its interaction with the endocytic scavenger receptor, low-density lipoprotein receptor-related protein 1 (LRP1). We discovered that suramin (C51H40N6O23S6) bound to TIMP-3 with a KD value of 1.9 ± 0.2 nM and inhibited its endocytosis via LRP1, thus increasing extracellular levels of TIMP-3 and inhibiting cartilage degradation by the TIMP-3 target enzyme, adamalysin-like metalloproteinase with thrombospondin motifs 5. NF279 (8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt), a structural analog of suramin, has an increased affinity for TIMP-3 and increased ability to inhibit TIMP-3 endocytosis and protect cartilage. Suramin is thus a promising scaffold for the development of novel therapeutics to increase TIMP-3 levels and inhibit cartilage degradation in osteoarthritis.

Generation of a mouse line harboring a Bi-transgene expressing luciferase and tamoxifen-activatable creER(T2) recombinase in cartilage.

We have used an aggrecan gene enhancer to generate a transgenic murine line (Acan-CreER-Ires-Luc) expressing firefly luciferase and tamoxifen activatable Cre recombinase (Cre-ER(T2) ). The expression and efficiency of the inducible Cre recombinase activity were tested in double transgenic mice created by crossing the Acan-CreER-Ires-Luc line with a Rosa26-lacZ reporter mouse. The expression pattern of the transgene of our line was restricted to cartilage from embryonic to adult stages. ß-galactosidase staining was observed in growth plate, articular cartilage, as well as fibrocartilage of meniscus, trachea, and intervertebral discs. Similar staining was observed in a previously described Agc1 (tm(IRES-creERT2)) murine line. The presence of luciferase in our transgene allows the visualization of the transgene expression in live animals. Weekly measurements from 2 to 8 weeks of age showed a reduction in luminescence in knee joints between 2 and 4 weeks of age, but stabilization thereafter. Following the surgical induction of osteoarthritis at 12 weeks of age, the level of luminescence remained the same in the knee joints for 8 weeks. This Acan-CreER-Ires-Luc murine line allows indirect monitoring of the transcriptional activity of the Acan gene via expression of luciferase, while the inducible Cre recombinase activity facilitates studies involving gain or loss of gene expression in cartilage.

Fibroblast growth factor 2 drives changes in gene expression following injury to murine cartilage in vitro and in vivo.

OBJECTIVE: The articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury. METHODS: Femoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2(-/-) mice. Reverse transcription-polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis. RESULTS: Murine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor-stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo. CONCLUSION: FGF-2-dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.

Joint immobilization prevents murine osteoarthritis and reveals the highly mechanosensitive nature of protease expression in vivo.

OBJECTIVE: Mechanical joint loading is critical for the development of osteoarthritis (OA). Although once regarded as a disease of cartilage attrition, OA is now known to be controlled by the expression and activity of key proteases, such as ADAMTS-5, that drive matrix degradation. This study was undertaken to investigate the link between protease expression and mechanical joint loading in vivo. METHODS: We performed a microarray analysis of genes expressed in the whole joint following surgical induction of murine OA (by cutting the medial meniscotibial ligament). Gene expression changes were validated by reverse transcriptase-polymerase chain reaction in whole joints and microdissected tissues of the joint, including the articular cartilage, meniscus, and epiphysis. Following surgery, mouse joints were immobilized, either by prolonged anesthesia or by sciatic neurectomy. RESULTS: Many genes were regulated in the whole joint within 6 hours of surgical induction of OA in the mouse. These included Arg1, Ccl2, Il6, Tsg6, Mmp3, Il1b, Adamts5, Adamts4, and Adamts1. All of these were significantly regulated in the articular cartilage. When joints were immobilized by prolonged anesthesia, regulation of the vast majority of genes was abrogated. When joints were immobilized by sciatic neurectomy, regulation of selected genes was abrogated, and OA was prevented up to 12 weeks postsurgery. CONCLUSION: These findings indicate that gene expression in the mouse joint following the induction of OA is rapid and highly mechanosensitive. Regulated genes include the known pathogenic protease ADAMTS-5. Targeting the mechanosensing mechanisms of joint tissue may offer new strategies for disease modification.

Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

Variants in ALDH1A2 reveal an anti-inflammatory role for retinoic acid and a new class of disease-modifying drugs in osteoarthritis.

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2, which encodes the key enzyme for synthesis of all-trans retinoic acid (atRA), are associated with severe hand OA. Here, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in the U.K. Biobank. Articular cartilage was acquired from 33 individuals with hand OA at the time of routine hand OA surgery. After stratification by genotype, RNA sequencing was performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulated similar inflammatory genes by a process that we have previously termed mechanoflammation, which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-inducible genes, which were used as a surrogate measure of cellular atRA concentration. Both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent mechanism. Talarozole was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6 hours after mouse knee joint destabilization and reduced cartilage degradation and osteophyte formation after 26 days. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo and identifies RAMBAs as potential disease-modifying drugs for OA.

2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Cyclic mechanical load causes global translational arrest in articular chondrocytes: a process which is partially dependent upon PKR phosphorylation.

The cellular mechanisms by which articular cartilage responds to load are poorly understood, but such responses may involve regulation at the level of protein translation rather than synthesis of mRNA. We investigated the role of translational control in cyclically (0.5 Hz, 0.1 Hz and 0.05 Hz) and statically loaded porcine articular cartilage explants. Messenger RNA was extracted for real time polymerase chain reaction (RT-PCR) and newly synthesised proteins were measured by their incorporation of radiolabelled 35S[methionine/cysteine] or 35SO4. Some medium from loaded and unloaded explants was immunoblotted for type II collagen, CTGF and TIMP3. The pathways that control protein translation were investigated by immunoblotting explant lysates for PKR, PERK (PKR like endoplasmic reticulum kinase), eIF2a (eukaryotic initiation factor 2a), eEFs (eukaryotic elongation factors), and AMP-dependent kinase. Explants were also loaded in the presence of inhibitors of PKR, the fibroblast growth factor (FGF) receptor and PI3 kinase. Cyclic loading caused complete global translational arrest as evidenced by a total suppression of new protein synthesis whilst maintaining mRNA levels. Translational arrest did not occur following static loading and was partly dependent upon the load frequency. There was a rebound increase in protein synthesis when labelling was performed after load had been withdrawn. Phosphorylation of PKR occurred in explants following cyclic load and inhibition of PKR modestly reversed suppression of newly synthesised proteins suggesting that PKR, at least in part, was responsible for loading induced translational arrest. These results show that translational control provides a rapid and potentially important mechanism for controlling the synthetic responses of articular chondrocytes in response to different types of mechanical load.

Deletions of immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia.

BACKGROUND: Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a haematopoietic stem cell. The hallmark genetic abnormality of CML is a chimeric BCR/ABL1 fusion gene resulting from the Philadelphia chromosome rearrangement t(9;22)(q34;q11). Clinical and laboratory studies indicate that the BCR/ABL1 fusion protein is essential for initiation, maintenance and progression of CML, yet the event(s) driving the transformation from chronic phase to blast phase are poorly understood. RESULTS: Here we report multiple genome aberrations in a collection of 78 CML and 14 control samples by oligonucleotide array comparative genomic hybridization. We found a unique signature of genome deletions within the immunoglobulin heavy chain (IGH) and T cell receptor regions (TCR), frequently accompanied by concomitant loss of sequences within the short arm regions of chromosomes 7 and 9, including IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2 genes. CONCLUSIONS: None of these genome losses were detected in any of the CML samples with myeloid transformation, chronic phase or controls, indicating that their presence is obligatory for the development of a malignant clone with a lymphoid phenotype. Notably, the coincidental deletions at IGH and TCR regions appear to precede the loss of IKZF1 and/or p16 genes in CML indicating a possible involvement of RAG in these deletions.

TSG-6 Is Weakly Chondroprotective in Murine OA but Does not Account for FGF2-Mediated Joint Protection.

OBJECTIVE: Tumor necrosis factor α-stimulated gene 6 (TSG-6) is an anti-inflammatory protein highly expressed in osteoarthritis (OA), but its influence on the course of OA is unknown. METHODS: Cartilage injury was assessed by murine hip avulsion or by recutting rested explants. Forty-two previously validated injury genes were quantified by real-time polymerase chain reaction in whole joints following destabilization of the medial meniscus (DMM) (6 hours and 7 days). Joint pathology was assessed at 8 and 12 weeks following DMM in 10-week-old male and female fibroblast growth factor 2 (FGF2)-/- , TSG-6-/- , TSG-6tg (overexpressing), FGF2-/- ;TSG-6tg (8 weeks only) mice, as well as strain-matched, wild-type controls. In vivo cartilage repair was assessed 8 weeks following focal cartilage injury in TSG-6tg and control mice. FGF2 release following cartilage injury was measured by enzyme-linked immunosorbent assay. RESULTS: TSG-6 messenger RNA upregulation was strongly FGF2-dependent upon injury in vitro and in vivo. Fifteeen inflammatory genes were significantly increased in TSG-6-/- joints, including IL1α, Ccl2, and Adamts5 compared with wild type. Six genes were significantly suppressed in TSG-6-/- joints including Timp1, Inhibin ßA, and podoplanin (known FGF2 target genes). FGF2 release upon cartilage injury was not influenced by levels of TSG-6. Cartilage degradation was significantly increased at 12 weeks post-DMM in male TSG-6-/- mice, with a nonsignificant 30% reduction in disease seen in TSG-6tg mice. No differences were observed in cartilage repair between genotypes. TSG-6 overexpression was unable to prevent accelerated OA in FGF2-/- mice. CONCLUSION: TSG-6 influences early gene regulation in the destabilized joint and exerts a modest late chondroprotective effect. Although strongly FGF2 dependent, TSG-6 does not explain the strong chondroprotective effect of FGF2.

Nociceptive Sensitizers Are Regulated in Damaged Joint Tissues, Including Articular Cartilage, When Osteoarthritic Mice Display Pain Behavior.

OBJECTIVE: Pain is the most common symptom of osteoarthritis (OA), yet where it originates in the joint and how it is driven are unknown. The aim of this study was to identify pain-sensitizing molecules that are regulated in the joint when mice subjected to surgical joint destabilization develop OA-related pain behavior, the tissues in which these molecules are being regulated, and the factors that control their regulation. METHODS: Ten-week-old mice underwent sham surgery, partial meniscectomy, or surgical destabilization of the medial meniscus (DMM). Pain-related behavior as determined by a variety of methods (testing of responses to von Frey filaments, cold plate testing for cold sensitivity, analgesiometry, incapacitance testing, and forced flexion testing) was assessed weekly. Once pain-related behavior was established, RNA was extracted from either whole joints or microdissected tissue samples (articular cartilage, meniscus, and bone). Reverse transcription-polymerase chain reaction analysis was performed to analyze the expression of 54 genes known to regulate pain sensitization. Cartilage injury assays were performed using avulsed immature hips from wild-type or genetically modified mice or by explanting articular cartilage from porcine joints preinjected with pharmacologic inhibitors. Levels of nerve growth factor (NGF) protein were measured by enzyme-linked immunosorbent assay. RESULTS: Mice developed pain-related behavior 8 weeks after undergoing partial meniscectomy or 12 weeks after undergoing DMM. NGF, bradykinin receptors B1 and B2, tachykinin, and tachykinin receptor 1 were significantly regulated in the joints of mice displaying pain-related behavior. Little regulation of inflammatory cytokines, leukocyte activation markers, or chemokines was observed. When tissue samples from articular cartilage, meniscus, and bone were analyzed separately, NGF was consistently regulated in the articular cartilage. The other pain sensitizers were also largely regulated in the articular cartilage, although there were some differences between the 2 models. NGF and tachykinin were strongly regulated by simple mechanical injury of cartilage in vitro in a transforming growth factor ß-activated kinase 1-, fibroblast growth factor 2-, and Src kinase-dependent manner. CONCLUSION: Damaged joint tissues produce proalgesic molecules, including NGF, in murine OA.

FISH mapping of Philadelphia negative BCR/ABL1 positive CML.

BACKGROUND: Chronic myeloid leukaemia (CML) is a haematopoietic stem cell disorder, almost always characterized by the presence of the Philadelphia chromosome (Ph), usually due to t(9;22)(q34;q11) or its variants. The Ph results in the formation of the BCR/ABL1 fusion gene, which is a constitutively activated tyrosine kinase. Around 1% of CML patients appear to have a Ph negative karyotype but carry a cryptic BCR/ABL1 fusion that can be located by fluorescence in situ hybridisation (FISH) at chromosome 22q11, 9q34 or a third chromosome. Here we present FISH mapping data of BCR and ABL1 flanking regions and associated chromosomal rearrangements in 9 Ph negative BCR/ABL1 positive CML patients plus the cell line CML-T1. RESULTS: BCR/ABL1 was located at 9q34 in 3 patients, 22q11 in 5 patients and CML-T1 and 22p11 in 1 patient. In 3 of 6 cases with the fusion at 22q11 a distal breakpoint cluster was found within a 280 Kb region containing the RAPGEF1 gene, while in another patient and the CML-T1 the distal breakpoint fell within a single BAC clone containing the 3' RXRA gene. Two cases had a duplication of the masked Ph while genomic deletions of the flanking regions were identified in 3 cases. Even more complex rearrangements were found in 3 further cases. CONCLUSION: BCR/ABL1 formation resulted from a direct insertion (one step mechanism) in 6 patients and CML-T1, while in 3 patients the fusion gene originated from a sequence of rearrangements (multiple steps). The presence of different rearrangements of both 9q34 and 22q11 regions highlights the genetic heterogeneity of this subgroup of CML. Future studies should be performed to confirm the presence of true breakpoint hot spots and assess their implications in Ph negative BCR/ABL1 positive CML.

Hypertrophic effects of urocortin homologous peptides are mediated via activation of the Akt pathway.

The UCN homologues SCP and SRP bind specifically to the CRFR2 receptor, whereas UCN binds to both CRFR1 and CRFR2. We have previously demonstrated that all three peptides are cardioprotective, and both the Akt and MAPK p42/44 pathways are essential for this effect. Here we tested the hypertrophic effects of these peptides. We examined the effects of the peptides on cell area, protein synthesis, and induction of the natriuretic peptides ANP and BNP. All three peptides were able to increase all the markers of hypertrophy examined, with SCP being the most potent of the three, followed by UCN and SRP last. In addition, we provide a mechanism of action for the three peptides and show that Akt phosphorylation is important for their hypertrophic action, whereas MAPK p42/44 is not involved in this effect.