Musclin (116-133) / Osteocrin (Ostn) (116-133) (Human)

Musclin (116-133) / Osteocrin (Ostn) (116-133) (Human) - FAM Labeled

	Catalog #	Size	Price
	FG-028-63A	1 nmol	$444

Sequence: ()

Phe-Gly-Ile-Pro-Met-Asp-Arg-Ile-Gly-Arg-Asn-Arg-Leu-Ser-Asn-Ser-Arg-Gly

Validation:	Exhibits correct molecular weight
Fluorescent Dye:	5(6)-Carboxyfluorescein (FAM)
Absorption:	495 nm Extinction Coefficient: 77400 M-1 cm-1
Max Emission:	519 nm
Contents:	Each vial contains 1 nmol of NET labeled peptide.

a Novel skeletal muscle-derived and Bone-specific Secreted Protein that Modulates the Osteoblast Phenotype

Alignments of musclin amino acid sequences for mouse, rat and human. Predicted signal sequence; shaded box. The putative serine protease cleavage site, KKKR; open box. The region homologous to mouse ANP, BNP and CNP; hatched box.

Predicted osteocrin protein structure and secretion.

A, predicted amino acid sequences from human, bovine, mouse, rat, chicken, and snake (partial) cDNAs. The two dibasic cleavage sites, 76KKKR79 and 110KKR112, are well conserved (boxed). Shaded residues represent non-conserved residues from the human sequence. Note that the C-terminal half of the protein is highly conserved.

B, immunofluorescent localization with a human osteocrin-specific antibody demonstrates overexpressed osteocrin is clearly visible in the secretory apparatus of HEK293A fibroblasts and UMR106 osteosarcoma cells (x400) colocalizing with the 58K Golgi protein-specific antibody.

C, the majority of osteocrin is detected in the medium of HEK293 cells by Western blot when transiently transfected (lanes 1 and 2). Residual protein in the secretory apparatus is detected in the cell lysate (lanes 4 and 5). Two exposures of the Western blot are shown to illustrate the underrepresented smaller processed fragments, with the lower panel representing a longer exposure. A doublet can be seen at the expected full-length size of 11.4 kDa and smaller processed fragments at ~ 5 kDa (lane 1). Mutation of the KKKR site to AS abolishes processing of the protein leaving only full-length 11.4 kDa bands (lane 2). Equal proportions of media or cell lysate from cultured cells were loaded. WT, wild type; Mut, mutated.

Osteocrin is a specific ligand of the natriuretic Peptide clearance receptor that modulates bone growth.

Osteocrin (Ostn) is a recently discovered secreted protein produced by cells of the osteoblast lineage that shows a well conserved homology with members of the natriuretic peptide (NP) family. We hypothesized that Ostn could interact with the NP receptors, thereby modulating NP actions on the skeleton. Ostn binds specifically and saturably to the NP peptide receptor-C (NPR-C) receptor with a Kd of approximately 5 nM with no binding to the GC-A or GC-B receptors. Deletion of several of the residues deemed important for NP binding to NPR-C led to abolition of Ostn binding, confirming the presence of a "natriuretic motif." Functionally, Ostn was able to augment C-type natriuretic peptide-stimulated cGMP production in both pre-chondrocytic (ATDC5) and osteoblastic (UMR106) cells, suggesting increased NP levels due to attenuation of NPR-C associated NP clearance. Ostn-transgenic mice displayed elongated bones and a marked kyphosis associated with elevated bone cGMP levels, suggesting that elevated natriuretic peptide activity contributed to the increased bone length possibly through an increase in growth plate chondrocyte proliferation. Thus, we have demonstrated that Ostn is a naturally occurring ligand of the NPR-C clearance receptor and may act to locally modulate the actions of the natriuretic system in bone by blocking the clearance action of NPR-C, thus locally elevating levels of C-type natriuretic peptide.

Moffatt P, Thomas G, Sellin K, et al. Osteocrin is a specific ligand of the natriuretic Peptide clearance receptor that modulates bone growth. J Biol Chem. 2007;282(50):36454-62.

Musclin, a novel skeletal muscle-derived secretory factor.

Skeletal muscle is involved in the homeostasis of glucose and lipid metabolism. We hypothesized that the skeletal muscle produces and secretes bioactive factor(s), similar to adipocytokines secreted by fat tissue. Here, we report the identification of a novel secretory factor, musclin, by signal sequence trap (SST) of mouse skeletal muscle cDNAs. Musclin cDNA encoded 130 amino acids, including N-terminal 30-amino acid signal sequence. Musclin protein contained a region homologous to natriuretic peptide (NP) family, and KKKR, a putative serine protease cleavage site, similar to NP family. Full-length musclin protein and KKKR-dependent cleaved form were secreted in media of musclin cDNA-transfected mammalian cell cultures. Musclin mRNA was expressed almost exclusively in the skeletal muscle of mice and rats. Musclin mRNA levels in skeletal muscle were markedly low in fasted, increased upon re-feeding, and were low in streptozotocin-treated insulin-deficient mice. Musclin mRNA expression was induced at late stage in the differentiation of C2C12 myocytes. In myocytes, insulin increased while epinephrine, isoproterenol and forskolin reduced musclin mRNA, all of which are known to increase the cellular content of cyclic AMP, a counter regulator to insulin. Pathologically, overexpression of musclin mRNA was noted in the muscles of obese insulin-resistant KKAy and db/db mice. Functionally, recombinant musclin significantly attenuated insulin-stimulated glucose uptake in myocytes. In conclusion, we identified musclin, a novel skeletal muscle-derived secretory factor. Musclin expression level is tightly regulated by nutritional changes and its physiological role could be linked to glucose metabolism.

Nishizawa H, Matsuda M, Yamada Y, et al. Musclin, a novel skeletal muscle-derived secretory factor. J Biol Chem. 2004;279(19):19391-5.

Ostn blocks the clearance action of NPR-C thereby increasing activity of the NPs in the bone compartment (A) Both the GC-B and NPR-C receptors are expressed in osteoblasts and chondrocytes, thus the activity of the NPs (normally CNP in the skeleton) is governed by the distribution of CNP between the signal mediating GC-B receptor and the NPR-C clearance receptor. (B) In the presence of Ostn, CNP access to NPR-C is blocked leading to increased binding of CNP to the GC-B receptor. This results in an increase in GC-B-mediated cGMP production, magnifying the downstream biological effects of the natriuretic system, which in the skeleton leads to elongated bones.

characterisation of binding of ostn to the npr-c receptor and its effects on ANP activity

Ostn-transgenic (Ostn-TG) mice have longer bones.

Ostn-TG mice with over-expression of Ostn in osteoblast lineage-specific cells were generated using the 3.6kb collagen type I promoter. Immunohistochemistry using an Ostn-specific antibody on 4-day-old tibia of wild type (WT) (A) and Ostn-TG (B) mice. Ostn expression in WT is non detectable (A) whereas overexpression in Ostn-TG is evident (red staining) on cuboidal osteoblasts adjacent to trabecular bone of the primary spongiosa (arrows), and within the perichondrium (arrowheads) (B). Counter stain is methylgreen. Scale bar is 200μm. (C, D) Gross appearance of WT (C) and Ostn-TG (D) which have a marked kyphosis presumably due to vertebral overgrowth. (E-F) Proliferation in growth plate chondrocytes was measured in tibiae of 7- week old male mice using PCNA immunohistochemistry. Proliferating chondrocytes are clearly more abundant in Ostn-TG (F) mice than WT littermates (E). Tail (G) and femur (H) lengths of 8-week old Ostn-TG line 650 males are significantly longer than wildtype littermates (n=7-12). (I) Quantification of PCNA immunohistochemistry showed an 80% increase in staining of growth plate chondrocytes (n=4) (J). cGMP levels were significantly higher in 10-14 day-old Ostn-TG mice femurs and tibiae than their WT littermates (n=11-28). *p<0.05; **p<0.01, Ostn-TG vs. WT. Data are expressed as mean ± SE. Comparison were made by ANOVA using Statview.

A, in situ localization of osteocrin transcripts in e16.5 mouse ribs. Osteocrin is localized within a subset of Cbfa-1-positive cells in a subperiosteal layer (per). Active mature osteoblasts are signified by an arrowhead (x200). B, expression of osteocrin in whole e17.5 fore limb. Osteocrin is clearly localized in the osteoblasts on the periphery of the bone (x100). C, in e16.5 mouse tibiae, osteocrin co-localizes with a subset of Cbfa-1-positive cells, exclusive from the more mature osteocalcin-positive cells found on the bone surface and in the trabecular bone (arrowheads) (x400).

A, Northern blot showing bone specificity of mouse osteocrin expression. Osteocrin is only detected in neonate and adult long bones and calvaria. No message was detected in embryonic (e) or adult (ad) non-bone tissues. Five µg total RNA was loaded. Br, brain; Go, gonads; Te, testes; He, heart; In, intestine; Ki, kidney; Li, liver; Lu, lung; Sp, spleen. B, osteocrin in detected by Northern blot in rat spleen. The transcript size of adult rat spleen osteocrin is smaller than that for adult rat calvaria. Fifteen µg of total RNA was loaded. The position of the 18 S ribosomal RNA band is marked. C, osteocrin expression was detected in embryonic calvariae and UMR106 cells by RT-PCR. No expression was detected in MG-63, SaoS-2, undifferentiated (-), or differentiated (+) MC3T3.E1 cells. D, Northern blot showing osteocrin expression in femora and calvariae in embryonic (e21), newborn (p4), growing (1 month, 1m), adult (3 month, 3m), and aged (8-month, 8m) rats. In femora, osteocrin expression was highest in newborn rats decreasing significantly in aged rats. Osteocrin expression peaks at 1-month in calvaria with a less marked decrease in older rats. Twenty-five µg of total RNA was loaded. ALP, alkaline phosphatase. For Figs. 4, A-D, GAPDH represents a loading control. E, immunohistochemistry with an osteocrin-specific antibody showing localization of osteocrin protein to active osteoblasts (Ob) in adult mouse tibia. The protein is absent from the mature osteocytes (Oc). No staining is visible in the sections stained with pre-immune serum (x400).

Osteocrin is expressed during osteoblast matrix production and maturation.

Northern blot of a time course of calvarial primary osteoblast differentiation at confluence and 5 and 10 days post-confluence in the presence and absence of 10 mM GP (PO4). Osteocrin is expressed at 5 and 10 days post-confluence but not at confluence. Cultures maintained in 10 mM GP until 10 days post-confluence exhibit a marked down-regulation in osteocrin expression. Osteocalcin and osteopontin expression are highest at 10 days post-confluence and are increased by GP treatment. Twenty µg of total RNA was loaded. GAPDH represents a loading control. Duplicate samples are shown.GP (PO4). Osteocrin is expressed at 5 and 10 days post-confluence but not at confluence. Cultures maintained in 10 mM GP until 10 days post-confluence exhibit a marked down-regulation in osteocrin expression. Osteocalcin and osteopontin expression are highest at 10 days post-confluence and are increased by GP treatment. Twenty µg of total RNA was loaded. GAPDH represents a loading control. Duplicate samples are shown.

Osteocrin regulates the osteoblast phenotype.

Primary calvarial osteoblastic cultures were treated with conditioned media from osteocrin or empty vector (control) transfected HEK293A cells from day 2 to 10 days post-confluence. A, 45Ca uptake was reduced 60% by osteocrin conditioned-media. Data is expressed as the mean ± S.E. of the % 45Ca incorporated into the cultures. *, p < 0.01. B, Northern blot demonstrating total repression of osteocalcin and a marked reduction in alkaline phosphatase expression in osteocrin treated cultures. Fifteen µg of total RNA was loaded. GAPDH represents a loading control. Duplicate samples are shown.

C, Secretion of musclin protein. The cell lysates and culture medium from C2C12 myocytes infected by retrovirus carrying Flag-tagged mouse musclin or GFP were immunoprecipitated, and subjected to western blot analysis, using anti-Flag antibody. Mus; musclin. D, Secretion of wild type and mutant musclin from HEK 293 cells. The culture medium from HEK 293 cells transfected with pEF-BOS-WT musclin Flag (left lane) or pEFBOS-Mut musclin Flag (76KKKR79-76A) (right lane) was subjected to immunoprecipitation followed by western blotting using anti-Flag antibody.

Regulation of musclin mRNA expression by nutritional status.

Musclin mRNA expression in insulin-deficient STZ mice.

In vivo expression of musclin mRNA. A, Northern blot analysis of musclin expression in mice. B, Quantitative RT-PCR analysis of musclin mRNA expression in mice. C, Regulation of musclin mRNA expression by nutritional status. Gastrocnemius muscles of mice fed ad libitum, fasted for 48 h, or fasted for 48 h and refed for 24 h (n=5, each) were used for real-time RT-PCR. Inset: representative Northern blotting (n=2, each). D, Musclin mRNA expression in insulin-deficient STZ mice. Gastrocnemius muscles of STZ and control mice (n=5, each) were subjected to real-time RT-PCR. The values were expressed relative to the level of 36B4 mRNA. E, Musclin mRNA expression in KKAy mice. Gastrocnemius muscles of 13-wk-old KKAy mice and age-matched C57BL/6J mice (n=8, each) were used for real-time RT-PCR. The values were normalized to the level of cyclophilin mRNA. Data are mean +- SEM values. *p< 0.05, **p< 0.01.

Regulation of musclin mRNA expression by various hormones, and effects of musclin on insulin-stimulated glucose uptake and glycogen synthesis in C2C12 myocytes. A, Musclin mRNA expression during myocyte differentiation. C2C12 cells were harvested at indicated days after differentiation-induction for real-time PCR. B-D, Regulation of musclin mRNA expression by insulin (INS), IGF-1 (IGF), epinephrine (Epi), isoproterenol (Iso), and forskolin (FSK) (real-time PCR). The expression level was expressed relative to that of cyclophlin mRNA (n=3, each). E-F, Effect of recombinant musclin on 2-DG uptake and glycogen synthesis. After 5-h pre-treatment with Flag-peptide (0.5 ug/ml) as control or Flagtagged musclin (0.5 ug/ml), 2-DG uptake (E) and glycogen synthesis (F) were determined as described in Experimental Procedures. Data are mean+- SEM values (n=6). **p<0.01. Similar results were obtained in two other independent experiments.

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