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Mactinin, a fragment of cytoskeletal alpha-actinin, is a novel inducer of heat shock protein (Hsp)-90 mediated monocyte activation.

Luikart SD, Panoskaltsis-Mortari A, Hinkel T, Perri RT, Gupta K, Oegema TR, Gupta P - BMC Cell Biol. (2009)

Bottom Line: Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD.Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex.Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNF-alpha, as well as monocyte chemotaxis).

View Article: PubMed Central - HTML - PubMed

Affiliation: Hematology/Oncology Section (111E), Veterans Affairs Medical Center, Minneapolis, MN 55417, USA. sharon.luikart@va.gov.

ABSTRACT

Background: Monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha and IL-1beta play important roles in cancer, inflammation, immune response and atherosclerosis. We previously showed that mactinin, a degradative fragment of the cytoskeletal protein alpha-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. We therefore sought to determine the mechanism by which mactinin stimulates monocytes.

Results: Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD. Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex. Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNF-alpha, as well as monocyte chemotaxis).

Conclusion: Mactinin is a novel inducer of Hsp90 activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." Blockage of this function of mactinin may be useful in diseases where monocyte/macrophage activation and/or Hsp90 activity are detrimental.

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Mactinin stimulates production of cytokines from monocytes. Human peripheral blood monocytes were incubated for 24 hrs with 100 nM mactinin, 100 nM α-actinin, 10 nM glutathione-S-transferase (GST), or no treatment. The concentrations of the indicated cytokines were determined in the supernatant. UD: undetectable at an assay sensitivity of 1.0 pg/ml. Data is shown as the mean +/- SEM. N = 3–4. Significance of differences between no treatment and mactinin: *P < 0.01.
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Figure 1: Mactinin stimulates production of cytokines from monocytes. Human peripheral blood monocytes were incubated for 24 hrs with 100 nM mactinin, 100 nM α-actinin, 10 nM glutathione-S-transferase (GST), or no treatment. The concentrations of the indicated cytokines were determined in the supernatant. UD: undetectable at an assay sensitivity of 1.0 pg/ml. Data is shown as the mean +/- SEM. N = 3–4. Significance of differences between no treatment and mactinin: *P < 0.01.

Mentions: Peripheral blood monocytes were isolated and cultured for 24 h with 100 nM mactinin, 100 nM α-actinin, 10 nM GST or medium alone (no treatment). The GST condition was included in order to control for the 10% contaminating GST in our mactinin preparation. Supernatants were recovered and centrifuged to remove nonadherent cells and aliquots assayed for the 3 cytokines. As shown in Fig. 1, the levels of IL-1α, IL-1β, and TNFα were significantly increased in the supernatants of mactinin-treated monocytes. Control cultures treated with α-actinin or GST did not show any increase in cytokine production. Mactinin did not stimulate the production of granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IL-12, macrophage colony-stimulating factor (M-CSF), or macrophage inhibitory protein (MIP)-1α (not shown). These findings indicate that mactinin directly stimulates the production of specific pro-inflammatory cytokines from monocytes.


Mactinin, a fragment of cytoskeletal alpha-actinin, is a novel inducer of heat shock protein (Hsp)-90 mediated monocyte activation.

Luikart SD, Panoskaltsis-Mortari A, Hinkel T, Perri RT, Gupta K, Oegema TR, Gupta P - BMC Cell Biol. (2009)

Mactinin stimulates production of cytokines from monocytes. Human peripheral blood monocytes were incubated for 24 hrs with 100 nM mactinin, 100 nM α-actinin, 10 nM glutathione-S-transferase (GST), or no treatment. The concentrations of the indicated cytokines were determined in the supernatant. UD: undetectable at an assay sensitivity of 1.0 pg/ml. Data is shown as the mean +/- SEM. N = 3–4. Significance of differences between no treatment and mactinin: *P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743639&req=5

Figure 1: Mactinin stimulates production of cytokines from monocytes. Human peripheral blood monocytes were incubated for 24 hrs with 100 nM mactinin, 100 nM α-actinin, 10 nM glutathione-S-transferase (GST), or no treatment. The concentrations of the indicated cytokines were determined in the supernatant. UD: undetectable at an assay sensitivity of 1.0 pg/ml. Data is shown as the mean +/- SEM. N = 3–4. Significance of differences between no treatment and mactinin: *P < 0.01.
Mentions: Peripheral blood monocytes were isolated and cultured for 24 h with 100 nM mactinin, 100 nM α-actinin, 10 nM GST or medium alone (no treatment). The GST condition was included in order to control for the 10% contaminating GST in our mactinin preparation. Supernatants were recovered and centrifuged to remove nonadherent cells and aliquots assayed for the 3 cytokines. As shown in Fig. 1, the levels of IL-1α, IL-1β, and TNFα were significantly increased in the supernatants of mactinin-treated monocytes. Control cultures treated with α-actinin or GST did not show any increase in cytokine production. Mactinin did not stimulate the production of granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IL-12, macrophage colony-stimulating factor (M-CSF), or macrophage inhibitory protein (MIP)-1α (not shown). These findings indicate that mactinin directly stimulates the production of specific pro-inflammatory cytokines from monocytes.

Bottom Line: Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD.Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex.Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNF-alpha, as well as monocyte chemotaxis).

View Article: PubMed Central - HTML - PubMed

Affiliation: Hematology/Oncology Section (111E), Veterans Affairs Medical Center, Minneapolis, MN 55417, USA. sharon.luikart@va.gov.

ABSTRACT

Background: Monocytes, their progeny such as dendritic cells and osteoclasts and products including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha and IL-1beta play important roles in cancer, inflammation, immune response and atherosclerosis. We previously showed that mactinin, a degradative fragment of the cytoskeletal protein alpha-actinin, is present at sites of monocytic activation in vivo, has chemotactic activity for monocytes and promotes monocyte/macrophage maturation. We therefore sought to determine the mechanism by which mactinin stimulates monocytes.

Results: Radiolabeled mactinin bound to a heterocomplex on monocytes comprised of at least 3 proteins of molecular weight 88 kD, 79 kD and 68 kD. Affinity purification, mass spectroscopy and Western immunoblotting identified heat shock protein (Hsp)-90 as the 88 kD component of this complex. Hsp90 was responsible for mediating the functional effects of mactinin on monocytes, since Hsp90 inhibitors (geldanamycin and its analogues 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG]) almost completely abrogated the stimulatory activity of mactinin on monocytes (production of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNF-alpha, as well as monocyte chemotaxis).

Conclusion: Mactinin is a novel inducer of Hsp90 activity on monocytes and may serve to perpetuate and augment monocytic activation, thereby functioning as a "matrikine." Blockage of this function of mactinin may be useful in diseases where monocyte/macrophage activation and/or Hsp90 activity are detrimental.

Show MeSH
Related in: MedlinePlus