Podosomes are highly dynamic actin-rich adhesion structures in cells of myeloid lineage and some transformed cells. Unlike transformed mesenchymal cell types, podosomes are the sole adhesion structure in macrophage and thus mediate all contact with adhesion substrate, including movement through complex tissues for immune surveillance. The existence of podosomes in inflammatory macrophages and transformed cell types suggest an important role in tissue invasion. The proteome, assembly, and maintenance of podosomes are emerging, but remain incompletely defined. Previously, we reported a formin homology sequence and actin assembly activity in association with macrophage β-3 integrin. In this study we demonstrate by quantitative RT-PCR and Western blotting that the formin FRL1 is specifically upregulated during monocyte differentiation to macrophages. We show that the formin FRL1 localizes to the actin-rich cores of primary macrophage podosomes. FRL1 co-precipitates with beta-3 integrin and both fixed and live cell fluorescence microscopy show that endogenous and overexpressed FRL1 selectively localize to macrophage podosomes. Targeted disruption of FRL1 by siRNA results in reduced cell adhesion and disruption of podosome dynamics. Our data suggest that FRL1 is responsible for modifying actin at the macrophage podosome and may be involved in actin cytoskeleton dynamics during adhesion and migration within tissues.

Formins are actin binding proteins conserved across species from plants to humans. The formin family is defined by their common FH2 domains. The 15 distinct human formins are involved in a broad range of cellular functions, including cell adhesion, cytokinesis, cell polarity, and cell morphogenesis. Their commonality is actin polymerization activity inherent to FH2 domains. While still requiring much study, biochemical activity of formins has been carefully described. In contrast, much less is known of their activities in complex living systems. With the diversity of the formin family and the actin structures that they affect, an extensive future of study beckons. In this study, we report the expression level of all 15 formins in 22 different human cell and tissue types using quantitative real-time PCR (qPCR). Identification of major themes in formin expression and documentation of expression profiles should facilitate the cellular study of formins.

No abstract

Prolactinomas are the most prevalent functional pituitary adenomas. Dopamine D2 receptor (D2R) agonists, such as bromocriptine are the first line of therapy; however, drug intolerance/resistance to D2R agonists exists. Apart from D2R agonists, there is no established medical therapy for prolactinomas; therefore, identifying novel therapeutics is warranted. Curcumin, a commonly used food additive in South Asian cooking, inhibits proliferation of several tumor cell lines; however, its effect on pituitary tumor cell proliferation has not been determined. Our objectives were to: 1) determine whether curcumin inhibits proliferation of pituitary tumor cell lines; 2) identify the signaling intermediaries that mediate the effect of curcumin; 3) examine whether curcumin inhibited pituitary hormone production and release; and 4) examine whether curcumin could enhance the growth-inhibitory effect of bromocriptine. Using rat lactotroph cell lines, GH3 and MMQ cells, we report that curcumin had a robust dose and time-dependent inhibitory effect on GH3 and MMQ cell proliferation. Inhibitory effects of curcumin persisted, even on removal of curcumin, and curcumin also blocked colony formation ability of pituitary tumor cells. The growth-inhibitory effect of curcumin was accompanied by decreased expression of cyclin D3 and ser 780 phosphorylation of retinoblastoma protein. In addition, curcumin also induced apoptosis in both GH3 and MMQ cells. Furthermore, curcumin suppresses intracellular levels and release of both prolactin and GH. Finally, we show that low concentrations of curcumin enhanced the growth-inhibitory effect of bromocriptine on MMQ cell proliferation. Taken together we demonstrate that curcumin inhibits pituitary tumor cell proliferation, induces apoptosis, and decreases hormone production and release, and thus, we propose developing curcumin as a novel therapeutic tool in the management of prolactinomas.

Having previously located the formin FMNL1 in macrophage podosomes, we developed an in vivo model to assess the role of FMNL1 in the migration activities of primary macrophages. Deletion of FMNL1 in mice was genetically lethal; however, targeted deletion in macrophages was achieved by employing macrophage-specific Cre. Unchallenged FMNL1-deficient mice exhibited an unexpected reduction in tissue-resident macrophages despite normal blood monocyte numbers. Upon immune stimulus, the absence of FMNL1 resulted in reduced macrophage recruitment in vivo, decreased migration in two-dimensional in vitro culture and a decrease in the number of macrophages exhibiting podosomes. Of the three described isoforms of FMNL1 -α, β and γ -only FMNL1γ rescued macrophage migration when expressed exogenously in depleted macrophages. Surprisingly, mutation of residues in the FH2 domain of FMNL1γ that disrupt barbed-end actin binding did not limit rescue of macrophage migration and podosome numbers. These observations suggest that FMNL1 contributes to macrophage migration activity by stabilizing the lifespan of podosomes without interaction of fast-growing actin termini.