Retinoids induce lumen morphogenesis in mammary epithelial cells

Abstract

Lumen formation is a fundamental step in the development of the structural and functional units of glandular organs, such as alveoli and ducts. In an attempt to elucidate the molecular signals that govern this morphogenetic event, we set up an in vitro system in which cloned mammary epithelial cells grown in collagen gels under serum-free conditions form solid, lumen-less colonies. Addition of as little as 0.1% donor calf serum (DCS) was sufficient to induce the formation of a central cavity. Among a number of serum constituents analyzed, retinol was found to mimic the effect of DCS in inducing lumen morphogenesis. Since the biological activities of retinol are largely dependent on its conversion to all-trans-retinoic acid (RA), we examined in more detail the effect of RA on lumen formation. RA induced the formation of lumen-containing colonies (cysts) in a concentration- and time-dependent manner, a half-maximal effect after 9 days of culture being observed with 100 pM RA. The pleiotropic effects of retinoids are mediated by nuclear retinoic acid receptors (RARs; α, β and γ) and retinoid X receptors (RXRs; α, β and γ). To identify the signaling pathway involved in RA-induced lumen formation, we used receptor-specific synthetic retinoids. TTNPB, a selective RAR agonist,promoted lumen morphogenesis, whereas RXR-selective ligands lacked this activity. Lumen formation was also induced at picomolar concentrations by Am-580, a synthetic retinoid that selectively binds the RARα receptor subtype. Moreover, co-addition of Ro 41-5253, an antagonist of RARα,abrogated the lumen-inducing activity of both RA and DCS, indicating that this biological response is mediated through an RARα-dependent signaling pathway. To gain insight into the mechanisms underlying RA-induced lumen formation, we assessed the potential role of matrix metalloproteinases (MMP). Using gelatin zymography, we observed a dose-dependent increase in latent and active forms of gelatinase B (MMP-9) upon RA treatment. In addition, lumen formation was abrogated by addition of the synthetic MMP inhibitor BB94,indicating that this morphogenetic process is likely to require MMP activity. Collectively, our results provide evidence that RA promotes lumen formation by mammary epithelial cells in vitro and suggest that it plays a similar role during mammary gland development in vivo.

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

“…An increase in MMP-9 (92 kDa gelatinase, gelatinase B) in mammary epithelial cells upon RA treatment, mediated through an RAR␣-dependent signaling pathway, has already been reported (24). To determine whether weaning modulates MMP-9 expression through the RAR␣ signaling pathway, real-time RT-PCR was performed in the mammary tissue from lactating and weaned rats.…”

“…Increased levels of MMP-2 and -3 during mammary gland involution. A: expression of MMP-2 and -3 mRNA in the mammary gland from C lactating rats, weaned rats (8,24, and 72 h, respectively), and WϩR rats was determined by real-time RT-PCR. Cyclophilin A was used as internal control (see MATERIALS AND METHODS).…”

“…Retinoids have been shown to modulate production of MMPs and TIMPs in mammary epithelial cells (24), endothelial cells, and alveolar macrophages (6,10). Numerous studies (23,32) in cell lines regarding the role of retinoids in breast cancer have been published.…”

Spatially restricted hyaluronan production byHas2drives epithelial tubulogenesis in vitro

“…J3B1A cells (34), a clonal derivative of the murine EpH4 mammary epithelial cell line (33), were grown in DMEM with Glutamax (Life Sciences, Bern, Switzerland) supplemented with 10% decomplemented donor calf serum (DCS; Life Sciences). Canine renal MDCK II Tet-Off cells were obtained from Clontech (Palo Alto, CA).…”

“…After a 10-min incubation at 37°C to allow collagen gelation, 0.5 ml of defined medium consisting of DMEM/F12, ITSϩ Premix (6.25 g/ml insulin, 6.25 g/ml transferrin, 6.25 ng/ml selenious acid, 1.25 mg/ml bovine serum albumin, and 5.35 g/ml linoleic acid; BD Biosciences, Bedford, MA), 2 ng/ml EGF (Pepro Tech), and 5 nM all-trans-retinoic acid (RA; Sigma) was added to the gels. Collagen gel cultures were then incubated in defined medium for 6 days to allow the formation of cystic structures (34). Thereafter, fresh defined medium was added with or without the indicated treatments.…”