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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).

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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-induced monocyte migration is blocked by Hsp90 Inhibitors. Monocytes were incubated with medium alone or with one of 3 Hsp90 inhibitors (G: geldanamycin; A: 17-AAG; D: 17-DMAG), prior to testing for mactinin-induced or fmpl-induced (control) chemotaxis. In the absence of mactinin or fmlp, 1302 +/- 122 cells migrated (not shown). Mactinin-induced migration was significantly inhibited by each of the Hsp90 inhibitors, whereas fmlp-induced migration was not. Data is shown as the mean +/- SEM. N = 3. Significance of differences in migration in the presence vs absence of Hsp90 inhibitors: *P < 0.01.
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Figure 3: Mactinin-induced monocyte migration is blocked by Hsp90 Inhibitors. Monocytes were incubated with medium alone or with one of 3 Hsp90 inhibitors (G: geldanamycin; A: 17-AAG; D: 17-DMAG), prior to testing for mactinin-induced or fmpl-induced (control) chemotaxis. In the absence of mactinin or fmlp, 1302 +/- 122 cells migrated (not shown). Mactinin-induced migration was significantly inhibited by each of the Hsp90 inhibitors, whereas fmlp-induced migration was not. Data is shown as the mean +/- SEM. N = 3. Significance of differences in migration in the presence vs absence of Hsp90 inhibitors: *P < 0.01.

Mentions: As shown in Fig. 3, all three Hsp90 inhibitors (geldanamycin, 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG], completely suppressed monocyte chemotaxis, since the number of cells that migrated towards mactinin in the presence of the Hsp90 inhibitors was comparable to the number of cells that migrated in medium alone (without mactinin; negative control). In contrast, fmlp-induced chemotaxis was not inhibited. The concentrations of the Hsp90 inhibitors tested included 100, 200, and 400 nM geldanamycin, 0.5 and 1 μM 17-AAG, and 100 nM 17-DMAG, based on previous reports of in vitro Hsp90 inhibition studies [29,30]. All concentrations significantly inhibited migration without meaningful differences (not shown).


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-induced monocyte migration is blocked by Hsp90 Inhibitors. Monocytes were incubated with medium alone or with one of 3 Hsp90 inhibitors (G: geldanamycin; A: 17-AAG; D: 17-DMAG), prior to testing for mactinin-induced or fmpl-induced (control) chemotaxis. In the absence of mactinin or fmlp, 1302 +/- 122 cells migrated (not shown). Mactinin-induced migration was significantly inhibited by each of the Hsp90 inhibitors, whereas fmlp-induced migration was not. Data is shown as the mean +/- SEM. N = 3. Significance of differences in migration in the presence vs absence of Hsp90 inhibitors: *P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Mactinin-induced monocyte migration is blocked by Hsp90 Inhibitors. Monocytes were incubated with medium alone or with one of 3 Hsp90 inhibitors (G: geldanamycin; A: 17-AAG; D: 17-DMAG), prior to testing for mactinin-induced or fmpl-induced (control) chemotaxis. In the absence of mactinin or fmlp, 1302 +/- 122 cells migrated (not shown). Mactinin-induced migration was significantly inhibited by each of the Hsp90 inhibitors, whereas fmlp-induced migration was not. Data is shown as the mean +/- SEM. N = 3. Significance of differences in migration in the presence vs absence of Hsp90 inhibitors: *P < 0.01.
Mentions: As shown in Fig. 3, all three Hsp90 inhibitors (geldanamycin, 17-allylamino-17-demethoxygeldanamycin [17-AAG] and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin [17-DMAG], completely suppressed monocyte chemotaxis, since the number of cells that migrated towards mactinin in the presence of the Hsp90 inhibitors was comparable to the number of cells that migrated in medium alone (without mactinin; negative control). In contrast, fmlp-induced chemotaxis was not inhibited. The concentrations of the Hsp90 inhibitors tested included 100, 200, and 400 nM geldanamycin, 0.5 and 1 μM 17-AAG, and 100 nM 17-DMAG, based on previous reports of in vitro Hsp90 inhibition studies [29,30]. All concentrations significantly inhibited migration without meaningful differences (not shown).

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