- Email: [email protected]
Reconsidering the Passive Diffusion Model of Steroid Hormone Cellular Entry
Developmental Cell
Previews Reconsidering the Passive Diffusion Model of Steroid Hormone Cellular Entry Sarah D. Neuman1 and Arash Bashirullah1,* 1Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.10.022
Steroid hormones have long been thought to enter target cells via passive diffusion through the plasma membrane. Now, reporting in Developmental Cell, Okamoto et al. (2018) demonstrate that, at least for Drosophila, steroid hormones require a protein transporter for cellular entry. Steroid hormones are unique among signaling molecules, as they must travel to the inside of their target cells to trigger biological responses. Once past the plasma membrane, steroid hormones bind to nuclear receptors, which are ligand-dependent transcription factors that directly regulate gene expression. In this way, steroid hormones influence nearly all aspects of animal development and physiology, including immunity, metabolism, growth, and sexual maturation. The mechanism by which steroid hormones enter their target cells has long been thought to be understood. Steroids are small, lipophilic molecules that are just ‘‘greasy’’ enough to enter phospholipid bilayers but also just ‘‘wet’’ enough to avoid becoming trapped within membranes. Given these characteristics, it seemed likely that steroid hormones could enter cells by passive diffusion across the plasma membrane, and indeed, several studies came to this very conclusion (Giorgi and Stein, 1981; Gorski and Gannon, 1976; Plagemann and Erbe, 1976). However, other studies suggested that steroid hormones require protein transporters for import into target cells, but no specific transporters were ever identified (Gorski and Gannon, 1976; Milgrom et al., 1973; Pietras and Szego, 1977). Thus, in the absence of a bona fide steroid hormone transporter, the simpler ‘‘passive diffusion’’ model prevailed for the last 40 years, so much so that many current biology and medical textbooks state this model as fact. However, in this issue of Developmental Cell, Okamoto et al. (2018) finally identify and characterize in the fruit fly Drosophila melanogaster a steroid hormone transporter protein, providing evidence that the prevailing dogma of steroid hormone
entry by passive diffusion needs to be reconsidered. Flies have one primary steroid hormone, called ecdysone, that drives each developmental transition throughout the fly life cycle. Previous work by members of this group demonstrated that ecdysone undergoes vesicle-mediated secretion from endocrine tissues, not passive diffusion through the membrane (Yamanaka et al., 2015). This discovery raised the tantalizing possibility that steroid hormone entry, like exit, may not occur via passive diffusion, suggesting that ecdysone may require a transporter for cellular import. To find this elusive, putative transporter, the authors conducted two separate genetic screens. The first was a candidate-based RNAi screen testing all possible transporter genes to identify those that disrupted ecdysone-dependent processes during fly development. The second was an unbiased, CRISPR-based forward genetic screen in Drosophila cell culture. This simple yet elegant two-pronged screening method identified a single gene, which they named Ecdysone Importer (EcI), that appeared to be required for ecdysone entry both in vivo and in vitro, highlighting the continued power of genetic screens to identify new genes and reveal new biology. EcI is an organic anion transporting polypeptide (OATP), part of the SLCO solute carrier superfamily of proteins that are widely conserved across animal species. OATPs transport small molecules across membranes; most notably, these proteins are known to facilitate transport of many human medications into cells (Hagenbuch and Stieger, 2013). OATPs contain 12 transmembrane domains and are thought to transport their cargoes through a single positively charged pore
(Kalliokoski and Niemi, 2009). The authors demonstrate that EcI exhibits the basic characteristics you would want to see in a steroid hormone transporter. EcI mRNA appeared to be ubiquitously expressed throughout development and among individual tissues. Furthermore, EcI was the primary OATP that was expressed in most fly tissues and the only OATP that was required for ecdysone function. Finally, EcI protein localized to the plasma membrane, placing this protein at the right time and place to function as a steroid hormone transporter. Functionally, EcI is required for ecdysone entry into cells. Steroid hormone signaling is indispensable for developmental progression. Accordingly, CRISPR-generated EcI mutant animals arrested development during early larval stages. Additionally, these mutant animals exhibited molting defects indicative of impaired ecdysone signaling. Several non-steroidal ecdysone mimetics have been identified (most are pesticides) (Nakagawa, 2005), and feeding EcI mutant larvae with one of these compounds, chromafenozide, rescued the developmental arrest phenotype, while feeding ecdysone did not. Chromafenozide is not a steroid; therefore, this small molecule likely enters cells independently of EcI. Since chromafenozide administration is sufficient to rescue the developmental arrest phenotype of EcI mutant animals, this result suggests that EcI is required for ecdysone import, but not for downstream ecdysone signaling. Additionally, the authors demonstrate that loss of EcI resulted in cell-autonomous disruption of ecdysone-induced transcriptional responses in vivo. Importantly, RNAi knockdown of EcI led to a cell-autonomous decrease in ecdysone
Developmental Cell 47, November 5, 2018 ª 2018 Elsevier Inc. 261
Developmental Cell
Previews titer, confirming that EcI is indeed necessary to import steroid hormones into cells. Finally, co-expression of EcI and the ecdysone receptor (EcR) in mammalian cells cultured in the presence of ecdysone activated expression of an EcRresponsive reporter, thereby convincingly demonstrating that EcI is both necessary and sufficient for ecdysone import into cells. So, how do steroid hormones enter cells? These findings by Okamoto and colleagues reopen a debate that was considered closed decades ago, as the identification and validation of a bona fide steroid hormone transporter now forces us to reconsider the validity of the passive diffusion model of steroid entry. From a biological perspective, transporter-mediated steroid hormone entry provides a new mechanism for cells to control how much, if any, steroid hormone they will import, adding an additional layer of regulation and complexity to steroid hormone signaling. However, the hydrophobicity profile of different steroid hormones varies considerably, making it difficult to directly extrapolate these findings with ecdysone to mammalian steroid hormones. Nevertheless, this study by Okamoto et al. (2018) creates reasonable doubt regarding the validity of the passive
diffusion model of steroid hormone entry, making it necessary to now determine whether transporters are required for entry of steroid hormones into mammalian cells. Given the wide evolutionary conservation of SLCO transporters among animals, it is possible that these proteins may also be required for steroid hormone uptake in mammalian cells. In fact, OATPs are already known to transport some steroid hormone precursors and conjugates in vivo, and these proteins have also been shown to transport steroid hormones in vitro (Hagenbuch and Stieger, 2013), raising the tantalizing possibility that careful in vivo studies may identify OATPs, and/or other transporter proteins, that are required for steroid hormone uptake in mammalian cells. If such transporters are confirmed, the implications for human health would be tremendous, as the presence of steroid hormone transporters would lead to revolutionary changes in the therapeutic approaches used to manage human endocrinerelated diseases.
Gorski, J., and Gannon, F. (1976). Current models of steroid hormone action: a critique. Annu. Rev. Physiol. 38, 425–450. Hagenbuch, B., and Stieger, B. (2013). The SLCO (former SLC21) superfamily of transporters. Mol. Aspects Med. 34, 396–412. Kalliokoski, A., and Niemi, M. (2009). Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. 158, 693–705. Milgrom, E., Atger, M., and Baulieu, E.-E. (1973). Studies on estrogen entry into uterine cells and on estradiol-receptor complex attachment to the nucleus–is the entry of estrogen into uterine cells a protein-mediated process? Biochim. Biophys. Acta 320, 267–283. Nakagawa, Y. (2005). Nonsteroidal ecdysone agonists. Vitam. Horm. 73, 131–173. Okamoto, N., Viswanatha, R., Bittar, R., Li, Z., Haga-Yamanaka, S., Perrimon, N., and Yamanaka, N. (2018). A membrane transporter is required for steroid hormone uptake in Drosophila. Dev. Cell 47, this issue, 294–305. Pietras, R.J., and Szego, C.M. (1977). Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265, 69–72. Plagemann, P.G.W., and Erbe, J. (1976). Glucocorticoids–uptake by simple diffusion by cultured Reuber and Novikoff rat hepatoma cells. Biochem. Pharmacol. 25, 1489–1494.
REFERENCES Giorgi, E.P., and Stein, W.D. (1981). The transport of steroids into animal cells in culture. Endocrinology 108, 688–697.
Yamanaka, N., Marque´s, G., and O’Connor, M.B. (2015). Vesicle-mediated steroid hormone secretion in Drosophila melanogaster. Cell 163, 907–919.
Locally Sourced: Auxin Biosynthesis and Transport in the Root Meristem Nicholas J. Morffy1 and Lucia C. Strader1,2,* 1Department
of Biology, Washington University, St. Louis, MO 63130, USA for Engineering MechanoBiology, Washington University, St. Louis, MO 63130, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.10.018 2Center
Localized maxima of the plant hormone auxin are crucial to root development and meristem maintenance. In this issue of Developmental Cell, Brumos et al. used elegant genetic and grafting experiments to distinguish between the contributions of local and distal auxin sources to auxin maxima generation and root meristem maintenance. Plants rely on a diverse array of endogenous signaling molecules to drive their development and responses to the environment. One of these signaling
molecules, auxin, impacts nearly every aspect of plant growth and development, including shoot growth, branching, and root growth (reviewed in Enders and
262 Developmental Cell 47, November 5, 2018 ª 2018 Elsevier Inc.
Strader, 2015). An auxin maximum is required for root meristem maintenance; auxin homeostasis models rely on the coordination of auxin biosynthesis, both
Previews Reconsidering the Passive Diffusion Model of Steroid Hormone Cellular Entry Sarah D. Neuman1 and Arash Bashirullah1,* 1Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.10.022
Steroid hormones have long been thought to enter target cells via passive diffusion through the plasma membrane. Now, reporting in Developmental Cell, Okamoto et al. (2018) demonstrate that, at least for Drosophila, steroid hormones require a protein transporter for cellular entry. Steroid hormones are unique among signaling molecules, as they must travel to the inside of their target cells to trigger biological responses. Once past the plasma membrane, steroid hormones bind to nuclear receptors, which are ligand-dependent transcription factors that directly regulate gene expression. In this way, steroid hormones influence nearly all aspects of animal development and physiology, including immunity, metabolism, growth, and sexual maturation. The mechanism by which steroid hormones enter their target cells has long been thought to be understood. Steroids are small, lipophilic molecules that are just ‘‘greasy’’ enough to enter phospholipid bilayers but also just ‘‘wet’’ enough to avoid becoming trapped within membranes. Given these characteristics, it seemed likely that steroid hormones could enter cells by passive diffusion across the plasma membrane, and indeed, several studies came to this very conclusion (Giorgi and Stein, 1981; Gorski and Gannon, 1976; Plagemann and Erbe, 1976). However, other studies suggested that steroid hormones require protein transporters for import into target cells, but no specific transporters were ever identified (Gorski and Gannon, 1976; Milgrom et al., 1973; Pietras and Szego, 1977). Thus, in the absence of a bona fide steroid hormone transporter, the simpler ‘‘passive diffusion’’ model prevailed for the last 40 years, so much so that many current biology and medical textbooks state this model as fact. However, in this issue of Developmental Cell, Okamoto et al. (2018) finally identify and characterize in the fruit fly Drosophila melanogaster a steroid hormone transporter protein, providing evidence that the prevailing dogma of steroid hormone
entry by passive diffusion needs to be reconsidered. Flies have one primary steroid hormone, called ecdysone, that drives each developmental transition throughout the fly life cycle. Previous work by members of this group demonstrated that ecdysone undergoes vesicle-mediated secretion from endocrine tissues, not passive diffusion through the membrane (Yamanaka et al., 2015). This discovery raised the tantalizing possibility that steroid hormone entry, like exit, may not occur via passive diffusion, suggesting that ecdysone may require a transporter for cellular import. To find this elusive, putative transporter, the authors conducted two separate genetic screens. The first was a candidate-based RNAi screen testing all possible transporter genes to identify those that disrupted ecdysone-dependent processes during fly development. The second was an unbiased, CRISPR-based forward genetic screen in Drosophila cell culture. This simple yet elegant two-pronged screening method identified a single gene, which they named Ecdysone Importer (EcI), that appeared to be required for ecdysone entry both in vivo and in vitro, highlighting the continued power of genetic screens to identify new genes and reveal new biology. EcI is an organic anion transporting polypeptide (OATP), part of the SLCO solute carrier superfamily of proteins that are widely conserved across animal species. OATPs transport small molecules across membranes; most notably, these proteins are known to facilitate transport of many human medications into cells (Hagenbuch and Stieger, 2013). OATPs contain 12 transmembrane domains and are thought to transport their cargoes through a single positively charged pore
(Kalliokoski and Niemi, 2009). The authors demonstrate that EcI exhibits the basic characteristics you would want to see in a steroid hormone transporter. EcI mRNA appeared to be ubiquitously expressed throughout development and among individual tissues. Furthermore, EcI was the primary OATP that was expressed in most fly tissues and the only OATP that was required for ecdysone function. Finally, EcI protein localized to the plasma membrane, placing this protein at the right time and place to function as a steroid hormone transporter. Functionally, EcI is required for ecdysone entry into cells. Steroid hormone signaling is indispensable for developmental progression. Accordingly, CRISPR-generated EcI mutant animals arrested development during early larval stages. Additionally, these mutant animals exhibited molting defects indicative of impaired ecdysone signaling. Several non-steroidal ecdysone mimetics have been identified (most are pesticides) (Nakagawa, 2005), and feeding EcI mutant larvae with one of these compounds, chromafenozide, rescued the developmental arrest phenotype, while feeding ecdysone did not. Chromafenozide is not a steroid; therefore, this small molecule likely enters cells independently of EcI. Since chromafenozide administration is sufficient to rescue the developmental arrest phenotype of EcI mutant animals, this result suggests that EcI is required for ecdysone import, but not for downstream ecdysone signaling. Additionally, the authors demonstrate that loss of EcI resulted in cell-autonomous disruption of ecdysone-induced transcriptional responses in vivo. Importantly, RNAi knockdown of EcI led to a cell-autonomous decrease in ecdysone
Developmental Cell 47, November 5, 2018 ª 2018 Elsevier Inc. 261
Developmental Cell
Previews titer, confirming that EcI is indeed necessary to import steroid hormones into cells. Finally, co-expression of EcI and the ecdysone receptor (EcR) in mammalian cells cultured in the presence of ecdysone activated expression of an EcRresponsive reporter, thereby convincingly demonstrating that EcI is both necessary and sufficient for ecdysone import into cells. So, how do steroid hormones enter cells? These findings by Okamoto and colleagues reopen a debate that was considered closed decades ago, as the identification and validation of a bona fide steroid hormone transporter now forces us to reconsider the validity of the passive diffusion model of steroid entry. From a biological perspective, transporter-mediated steroid hormone entry provides a new mechanism for cells to control how much, if any, steroid hormone they will import, adding an additional layer of regulation and complexity to steroid hormone signaling. However, the hydrophobicity profile of different steroid hormones varies considerably, making it difficult to directly extrapolate these findings with ecdysone to mammalian steroid hormones. Nevertheless, this study by Okamoto et al. (2018) creates reasonable doubt regarding the validity of the passive
diffusion model of steroid hormone entry, making it necessary to now determine whether transporters are required for entry of steroid hormones into mammalian cells. Given the wide evolutionary conservation of SLCO transporters among animals, it is possible that these proteins may also be required for steroid hormone uptake in mammalian cells. In fact, OATPs are already known to transport some steroid hormone precursors and conjugates in vivo, and these proteins have also been shown to transport steroid hormones in vitro (Hagenbuch and Stieger, 2013), raising the tantalizing possibility that careful in vivo studies may identify OATPs, and/or other transporter proteins, that are required for steroid hormone uptake in mammalian cells. If such transporters are confirmed, the implications for human health would be tremendous, as the presence of steroid hormone transporters would lead to revolutionary changes in the therapeutic approaches used to manage human endocrinerelated diseases.
Gorski, J., and Gannon, F. (1976). Current models of steroid hormone action: a critique. Annu. Rev. Physiol. 38, 425–450. Hagenbuch, B., and Stieger, B. (2013). The SLCO (former SLC21) superfamily of transporters. Mol. Aspects Med. 34, 396–412. Kalliokoski, A., and Niemi, M. (2009). Impact of OATP transporters on pharmacokinetics. Br. J. Pharmacol. 158, 693–705. Milgrom, E., Atger, M., and Baulieu, E.-E. (1973). Studies on estrogen entry into uterine cells and on estradiol-receptor complex attachment to the nucleus–is the entry of estrogen into uterine cells a protein-mediated process? Biochim. Biophys. Acta 320, 267–283. Nakagawa, Y. (2005). Nonsteroidal ecdysone agonists. Vitam. Horm. 73, 131–173. Okamoto, N., Viswanatha, R., Bittar, R., Li, Z., Haga-Yamanaka, S., Perrimon, N., and Yamanaka, N. (2018). A membrane transporter is required for steroid hormone uptake in Drosophila. Dev. Cell 47, this issue, 294–305. Pietras, R.J., and Szego, C.M. (1977). Specific binding sites for oestrogen at the outer surfaces of isolated endometrial cells. Nature 265, 69–72. Plagemann, P.G.W., and Erbe, J. (1976). Glucocorticoids–uptake by simple diffusion by cultured Reuber and Novikoff rat hepatoma cells. Biochem. Pharmacol. 25, 1489–1494.
REFERENCES Giorgi, E.P., and Stein, W.D. (1981). The transport of steroids into animal cells in culture. Endocrinology 108, 688–697.
Yamanaka, N., Marque´s, G., and O’Connor, M.B. (2015). Vesicle-mediated steroid hormone secretion in Drosophila melanogaster. Cell 163, 907–919.
Locally Sourced: Auxin Biosynthesis and Transport in the Root Meristem Nicholas J. Morffy1 and Lucia C. Strader1,2,* 1Department
of Biology, Washington University, St. Louis, MO 63130, USA for Engineering MechanoBiology, Washington University, St. Louis, MO 63130, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.10.018 2Center
Localized maxima of the plant hormone auxin are crucial to root development and meristem maintenance. In this issue of Developmental Cell, Brumos et al. used elegant genetic and grafting experiments to distinguish between the contributions of local and distal auxin sources to auxin maxima generation and root meristem maintenance. Plants rely on a diverse array of endogenous signaling molecules to drive their development and responses to the environment. One of these signaling
molecules, auxin, impacts nearly every aspect of plant growth and development, including shoot growth, branching, and root growth (reviewed in Enders and
262 Developmental Cell 47, November 5, 2018 ª 2018 Elsevier Inc.
Strader, 2015). An auxin maximum is required for root meristem maintenance; auxin homeostasis models rely on the coordination of auxin biosynthesis, both