SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland
2016; Springer Nature; Volume: 36; Issue: 2 Linguagem: Inglês
10.15252/embj.201694387
ISSN1460-2075
AutoresEmilia Peuhu, Riina Kaukonen, Martina Lerche, Markku Saari, Camilo Guzmán, Pia Rantakari, Nicola De Franceschi, Anni Wärri, Μαρία Γεωργιάδου, Guillaume Jacquemet, Elina Mattila, Reetta Virtakoivu, Yuming Liu, Youmna Attieh, Kathleen A. Silva, Timo Betz, John P. Sundberg, Marko Salmi, Marie‐Ange Deugnier, Kevin W. Eliceiri, Johanna Ivaska,
Tópico(s)Bone and Dental Protein Studies
ResumoArticle14 December 2016free access Transparent process SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland Emilia Peuhu Corresponding Author Emilia Peuhu [email protected] orcid.org/0000-0003-4945-3046 Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Riina Kaukonen Riina Kaukonen Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Martina Lerche Martina Lerche Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Markku Saari Markku Saari Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Camilo Guzmán Camilo Guzmán Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Pia Rantakari Pia Rantakari orcid.org/0000-0003-1638-5075 MediCity Research Laboratory, University of Turku, Turku, Finland Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland Search for more papers by this author Nicola De Franceschi Nicola De Franceschi Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Anni Wärri Anni Wärri Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Maria Georgiadou Maria Georgiadou Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Guillaume Jacquemet Guillaume Jacquemet Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Elina Mattila Elina Mattila Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Reetta Virtakoivu Reetta Virtakoivu Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Yuming Liu Yuming Liu orcid.org/0000-0001-5391-8892 Department of Biomedical Engineering, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin at Madison, Madison, WI, USA Search for more papers by this author Youmna Attieh Youmna Attieh Institut Curie, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Kathleen A Silva Kathleen A Silva The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Timo Betz Timo Betz Institut Curie, Paris Sciences et Lettres Research University, Paris, France Center for Molecular Biology of Inflammation, Cells-in-Motion Cluster of Excellence, Institute of Cell Biology, Münster University, Münster, Germany Search for more papers by this author John P Sundberg John P Sundberg The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Marko Salmi Marko Salmi MediCity Research Laboratory, University of Turku, Turku, Finland Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland Search for more papers by this author Marie-Ange Deugnier Marie-Ange Deugnier Institut Curie, Paris Sciences et Lettres Research University, Paris, France Institut Curie, CNRS, UMR144, Paris, France Search for more papers by this author Kevin W Eliceiri Kevin W Eliceiri Department of Biomedical Engineering, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin at Madison, Madison, WI, USA Search for more papers by this author Johanna Ivaska Corresponding Author Johanna Ivaska [email protected] orcid.org/0000-0002-6295-6556 Centre for Biotechnology, University of Turku, Turku, Finland Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Emilia Peuhu Corresponding Author Emilia Peuhu [email protected] orcid.org/0000-0003-4945-3046 Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Riina Kaukonen Riina Kaukonen Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Martina Lerche Martina Lerche Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Markku Saari Markku Saari Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Camilo Guzmán Camilo Guzmán Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Pia Rantakari Pia Rantakari orcid.org/0000-0003-1638-5075 MediCity Research Laboratory, University of Turku, Turku, Finland Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland Search for more papers by this author Nicola De Franceschi Nicola De Franceschi Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Anni Wärri Anni Wärri Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Maria Georgiadou Maria Georgiadou Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Guillaume Jacquemet Guillaume Jacquemet Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Elina Mattila Elina Mattila Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Reetta Virtakoivu Reetta Virtakoivu Centre for Biotechnology, University of Turku, Turku, Finland Search for more papers by this author Yuming Liu Yuming Liu orcid.org/0000-0001-5391-8892 Department of Biomedical Engineering, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin at Madison, Madison, WI, USA Search for more papers by this author Youmna Attieh Youmna Attieh Institut Curie, Paris Sciences et Lettres Research University, Paris, France Search for more papers by this author Kathleen A Silva Kathleen A Silva The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Timo Betz Timo Betz Institut Curie, Paris Sciences et Lettres Research University, Paris, France Center for Molecular Biology of Inflammation, Cells-in-Motion Cluster of Excellence, Institute of Cell Biology, Münster University, Münster, Germany Search for more papers by this author John P Sundberg John P Sundberg The Jackson Laboratory, Bar Harbor, ME, USA Search for more papers by this author Marko Salmi Marko Salmi MediCity Research Laboratory, University of Turku, Turku, Finland Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland Search for more papers by this author Marie-Ange Deugnier Marie-Ange Deugnier Institut Curie, Paris Sciences et Lettres Research University, Paris, France Institut Curie, CNRS, UMR144, Paris, France Search for more papers by this author Kevin W Eliceiri Kevin W Eliceiri Department of Biomedical Engineering, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin at Madison, Madison, WI, USA Search for more papers by this author Johanna Ivaska Corresponding Author Johanna Ivaska [email protected] orcid.org/0000-0002-6295-6556 Centre for Biotechnology, University of Turku, Turku, Finland Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland Search for more papers by this author Author Information Emilia Peuhu *,1, Riina Kaukonen1,‡, Martina Lerche1,‡, Markku Saari1, Camilo Guzmán1, Pia Rantakari2,3, Nicola De Franceschi1, Anni Wärri1, Maria Georgiadou1, Guillaume Jacquemet1, Elina Mattila1, Reetta Virtakoivu1, Yuming Liu4, Youmna Attieh5, Kathleen A Silva6, Timo Betz5,7, John P Sundberg6, Marko Salmi2,3, Marie-Ange Deugnier5,8, Kevin W Eliceiri4 and Johanna Ivaska *,1,9 1Centre for Biotechnology, University of Turku, Turku, Finland 2MediCity Research Laboratory, University of Turku, Turku, Finland 3Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland 4Department of Biomedical Engineering, Laboratory for Optical and Computational Instrumentation (LOCI), University of Wisconsin at Madison, Madison, WI, USA 5Institut Curie, Paris Sciences et Lettres Research University, Paris, France 6The Jackson Laboratory, Bar Harbor, ME, USA 7Center for Molecular Biology of Inflammation, Cells-in-Motion Cluster of Excellence, Institute of Cell Biology, Münster University, Münster, Germany 8Institut Curie, CNRS, UMR144, Paris, France 9Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland ‡These authors contributed equally to this work *Corresponding author. Tel: +358 2 333 7953; E-mail: [email protected] *Corresponding author. Tel: +358 2 333 7954; E-mail: [email protected] The EMBO Journal (2017)36:165-182https://doi.org/10.15252/embj.201694387 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpincpdm), and mice with a stromal (S100a4-Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpincpdm mammary epithelial cells transplanted in vivo into wild-type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpincpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpincpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM. Synopsis SHARPIN is implicated in regulation of extracellular matrix adhesion molecules. New data show that SHARPIN modulates stromal collagen architecture and matrix turnover in mammary gland fibroblasts during puberty and thereby regulates the outgrowth of mammary ducts. Mice lacking SHARPIN expression (Sharpincpdm) have reduced mammary ductal outgrowth during puberty. Transplantation studies and conditional knockouts show that stromal, and not epithelial, SHARPIN regulates mammary gland development. Collagen bundling and extracellular matrix stiffness are reduced in the Sharpincpdm mammary gland stroma. Mammary gland fibroblasts that lack SHARPIN demonstrate defective collagen remodelling and turnover in vitro. Introduction The mammary gland develops post-natally in response to growth and steroid hormones, and local growth factors. During mammary ductal elongation and branching morphogenesis, the pubertal mammary epithelium invades into the fat pad stroma to form the gland that later evolves further during the menstrual cycle, and terminally differentiates/dedifferentiates during pregnancy, lactation and involution. Mammary ductal outgrowth takes place at the tips of the ducts, in the terminal end buds (TEBs). Here, in areas of active cell division, hollow ducts are formed through luminal apoptosis, and cells undergo differentiation into luminal and basal mammary epithelial layers (Hinck & Silberstein, 2005; Ewald et al, 2008). Complex signalling between the epithelium and the stroma orchestrates the mammary ductal outgrowth and branching through the adipose tissue (Sternlicht et al, 2006; Howard & Lu, 2014). This process involves significant regulation of the surrounding extracellular matrix (ECM) (Zhu et al, 2014; Gomes et al, 2015). Epithelial cell adhesion to the surrounding ECM via integrins, heterodimeric transmembrane adhesion receptors, plays an important role in mammary ductal outgrowth (Klinowska et al, 1999), in preserving the regenerative capacity of the mammary epithelium (Taddei et al, 2008), and during breast cancer invasion and metastasis (Levental et al, 2009). However, the signalling pathways that regulate mammary gland stromal cell adhesion and collagen architecture, and thereby ductal outgrowth, are largely unknown. SHARPIN (Shank-associated RH domain-interacting protein, also known as SIPL1) binds to intracellular integrin alpha tails of inactive integrins and inhibits recruitment of talin and kindlin to the beta tail, thereby functioning as an integrin inhibitor (Rantala et al, 2011; Pouwels et al, 2013; De Franceschi et al, 2015). SHARPIN is also an essential component of the linear ubiquitination assembly complex (LUBAC) (Gerlach et al, 2011; Ikeda et al, 2011; Tokunaga et al, 2011) that regulates canonical nuclear factor-κB (NF-κB) signalling in response to cytokines, bacteria and genotoxic stress through linear ubiquitination (Gerlach et al, 2011; Tokunaga et al, 2011; Fujita et al, 2014). Furthermore, SHARPIN is involved in negative regulation of T-cell receptor signalling (Park et al, 2016), priming of the NLRP3 inflammasome complex in macrophages (Rodgers et al, 2014; Gurung et al, 2015), and it has been reported to bind and regulate key signalling proteins such as eyes absent homolog 1 (EYA1) (Landgraf et al, 2010), SH3 and multiple ankyrin repeat domains protein (SHANK) (Lim et al, 2001), and phosphatase and tensin homolog (PTEN) (He et al, 2010). SHARPIN-deficient (Sharpincpdm) mice display defective secondary lymphoid organ development (HogenEsch et al, 1993; Seymour et al, 2013) and suffer from progressive multiorgan inflammation with chronic eosinophilic hyperproliferative dermatitis due to increased tumour necrosis factor receptor (TNFR)-mediated keratinocyte apoptosis (Seymour et al, 2007; Gerlach et al, 2011; Rickard et al, 2014). Increased SHARPIN expression has previously been linked to prostate tumorigenesis (He et al, 2010; Li et al, 2015), elevated breast cancer risk (De Melo & Tang, 2015) and breast cancer metastasis (Bii et al, 2015), suggesting a role for SHARPIN in regulating epithelial homeostasis. Many of the molecular features driving invasive breast carcinoma are also essential during normal mammary ductal outgrowth, including collective cell migration, ECM remodelling and epithelial–stromal communication (Polyak & Kalluri, 2010). The potential involvement of SHARPIN in processes related to breast cancer invasion and metastasis prompted us to investigate post-natal mammary gland development in Sharpincpdm mice. Here, we report that Sharpincpdm mice have defective mammary ductal outgrowth during puberty and demonstrate an epithelial cell extrinsic requirement for SHARPIN in regulating normal stromal collagen architecture and stiffness. Accordingly, Sharpincpdm fibroblasts demonstrate an inability to generate traction forces on collagen and to assemble, contract and degrade collagen fibres. Results SHARPIN is expressed in the mammary gland To examine the expression of SHARPIN in the mammary gland, paraffin-embedded human tissue sections were stained by immunohistochemistry (IHC) (Fig 1A). SHARPIN expression was detected in the luminal epithelial cell layer and in the scattered stromal cells, but not in the basal epithelial cells directly adhering to the basal lamina (Fig 1A). Co-staining of SHARPIN with vimentin confirmed that the majority of the SHARPIN-positive stromal cells were spindle-shaped and vimentin expressing fibroblasts (Fig EV1A). For further characterisation, mouse mammary gland epithelial cells (MECs) and mammary gland stromal fibroblasts (MSFs) were isolated, and the expression of SHARPIN was analysed by Western blotting (Fig 1B). SHARPIN was expressed at the protein level in both mammary gland primary cell populations although more prominently in the epithelial portion (Fig 1B). The specific expression of CDH1 (also called E-cadherin), detected as a double band (upper band represents the unprocessed receptor form) (Fujita et al, 2002), in the epithelial cells and vimentin in the stromal cells confirmed the purity of the two populations (Fig 1B). MECs and MSFs were further sorted by flow cytometry to basal epithelial, luminal epithelial (progenitors/mature) and stromal populations based on CD24 and ICAM1 surface expressions (Di-Cicco et al, 2015; Fig 1C). Lineage-specific marker mRNA expression [basal, keratin 5 (Krt5); luminal, keratin 18 (Krt18); stromal, Pdgfra] was controlled by quantitative PCR (qPCR) (Fig EV1B). Sharpin mRNA expression was low in basal epithelial cells (LinnegCD24intICAM1hi), higher in luminal progenitor (LinnegCD24hi ICAM1int) and mature luminal epithelial cells (LinnegCD24hi ICAM1neg) and highest in stromal fibroblasts (LinnegCD24neg) when measured by qPCR (Fig 1D). Taken together, our results show that SHARPIN mRNA and protein are expressed both in the epithelial and in the stromal cells of the mouse mammary gland. Figure 1. SHARPIN is expressed in the stromal and luminal epithelial cells of the mammary gland Immunohistochemical analysis of SHARPIN expression in the human mammary gland. Cross section of a mammary duct (upper panel) and magnification of the marked area (lower panel). SHARPIN-positive luminal (grey arrow) and stromal cells (red arrow), and the approximate position of the basal lamina (dashed red line) are indicated. Scale bar represents 50 μm. Western blot analysis of SHARPIN protein expression in isolated primary mammary epithelial cells (MECs) and mammary stromal fibroblasts (MSFs). CDH1 and vimentin were used as markers of epithelial and stromal cell lineages, respectively. GAPDH served as a control for protein loading. FACS-based isolation of mouse mammary gland basal epithelial cells (LinnegCD24intICAM-1hi), mature luminal epithelial cells (LinnegCD24hiICAM-1neg), luminal progenitor cells (LinnegCD24hiICAM-1int) and stromal cells (LinnegCD24neg). Quantitative PCR analysis of Sharpin mRNA expression in cell populations isolated in (C) (mean ± SEM, n = 3–5). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. SHARPIN expression in the mammary gland cells Co-staining of SHARPIN and vimentin in stromal fibroblasts of human breast tissue. Upper panel scale bar 50 μm. Magnification shown in lower panel, scale bar 20 μm. Expression of lineage-specific marker genes at RNA level (basal, Krt5; luminal, Krt18; stromal, Pdgfra) was measured by qPCR from FACS-sorted mammary gland basal, luminal and stromal cell populations in Fig 1D (n = 3). Mean ± SEM. Download figure Download PowerPoint SHARPIN-deficient mammary glands exhibit reduced ductal outgrowth during puberty Mammary ductal morphogenesis in pubertal female mice has been previously investigated to identify mechanisms potentially hijacked by breast cancer cells for adhesion, invasion and metastasis (Lanigan et al, 2007; Polyak & Kalluri, 2010). As SHARPIN is expressed in the mammary gland (Fig 1) and is known to regulate both cell adhesion (Rantala et al, 2011) and breast cancer metastasis (Bii et al, 2015; De Melo & Tang, 2015), we hypothesised that SHARPIN could play an important role during mammary ductal outgrowth. To examine this, the 4th mammary glands were isolated from 3- to 7-week-old female wt and SHARPIN-deficient (Sharpincpdm) mice (Figs 2A and EV2A), and ductal outgrowth and branching were quantified from carmine alum-stained whole mounts (Fig 2B and C). While the ductal trees of the pre-pubertal 3- to 4-week-old mice were equal in size, a clear reduction in ductal outgrowth area was observed in the Sharpincpdm mammary glands at puberty (5–7 weeks old; Fig 2A and B), indicating impaired pubertal (allometric) mammary growth. Additionally, the number of ductal branches per gland was significantly lower in pubertal Sharpincpdm mice (Fig 2C). These differences were not attributed to disturbances in the onset of puberty in the Sharpincpdm mice, as it occurred normally close to 5 weeks of age similarly to their wt female littermate controls, as judged based on the evaluation of vaginal opening (Fig EV2B). Furthermore, oestrogen receptor and progesterone receptor expressions were similar in both wt and Sharpincpdm mammary glands indicative of normal systemic steroid hormone production at puberty (Fig EV2C). The polarity of the mammary ductal cell layers was also similar in wt and Sharpincpdm mice as examined by hematoxylin-eosin (HE) and IHC labelling of luminal (CDH1) and basal (integrin alpha 6; ITGA6) epithelial cells from histological sections of 7-week-old mouse mammary glands (Fig 2D). Figure 2. Mammary ductal outgrowth during puberty is impaired in SHARPIN-deficient (Sharpincpdm) miceMammary ductal outgrowth in 3- to 7-week-old wt or Sharpincpdm female mice. A. Representative carmine alum-stained mammary gland whole mounts. Arrow indicates the inguinal lymph node. Scale bars represent 2 mm. B. Quantification of mammary ductal outgrowth area (n = 4–15 glands). C. Quantification of the number of ductal branch points per mammary gland (n = 7–9 glands, 7-week-old mice). D. Cryosections from 7-week-old wt or Sharpincpdm mouse mammary glands stained with hematoxylin-eosin (HE) (upper panel) or immunolabelled with the indicated antibodies. Scale bars represent 20 μm. E, F. (E) Representative carmine alum-stained images highlighting terminal end buds (TEBs) in 7-week-old wt and Sharpincpdm mouse mammary glands and (F) quantification of the number of TEBs per gland (n = 9–10 glands). Scale bar represents 500 μm. G. TEBs in paraffin sections from wt or Sharpincpdm mouse mammary glands stained with HE (upper panel) or immunolabelled with the indicated antibodies (lower panel). Scale bars represent 50 μm. Data information: (B, C, F) Mean ± SEM. (B) Unpaired Student's t-test or Mann–Whitney test, (C) Mann–Whitney test. (F) Unpaired Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Mammary epithelial SHARPIN is dispensable for pubertal ductal invasion SHARPIN protein expression in primary mouse mammary epithelial cells (MEC) and mammary stromal fibroblasts (MSF) isolated from wt and Sharpincpdm mice. GAPDH expression was used for measurement of protein loading. Puberty onset determined by the first day of vaginal opening (mean ± SEM, wt n = 9; Sharpincpdm n = 4, Mann–Whitney test). Immunohistochemistry (IHC) of oestrogen receptor (ESR1, upper panel, n = 4 mice) and progesterone receptor (PGR, lower panel, n = 2 mice) expressions in pubertal wt and Sharpincpdm mouse mammary glands at 6–7 weeks of age. KRT8 and ACTA2 were co-labelled with PGR to identify mammary ducts. Scale bar 20 μm. Percentage of positively stained nuclear area within epithelium was quantified (mean ± SEM). Paraffin sections from BrdU-injected (200 μl/20 g i.p. 2 h before sacrifice) or non-injected 7-week-old wt and Sharpincpdm mice were labelled with BrdU antibody (upper panel; n = 2 mice) or with ACTA2 and cleaved caspase-3 (CASP3) antibodies (lower panel; n = 3 mice). Scale bar 20 μm. Positively stained cells per 0.1 mm2 epithelium were scored (mean ± SEM). Flow cytometric analysis of MEC subpopulations in virgin wt and Sharpincpdm mice; basal epithelial cells: LinnegCD24intCD29+ or LinnegCD24intCD49f+; luminal epithelial cells: LinnegCD24hiCD29neg or LinnegCD24hiCD49fneg. The results are representative of three independent experiments. Sharpincpdm MECs undergo normal tertiary branching when transplanted in wt host fat pads. Quantification of ductal branching in pregnant (P15) wt and Sharpincpdm mammary epithelial transplants in wt hosts (n = 5 mice) in ImageJ software (horizontal line, median; box, 25–75th percentile; whiskers, 10–90th percentile; unpaired Student's t-test). Download figure Download PowerPoint As TEBs of the mammary ductal epithelium are known to be responsible for ductal growth and invasion through the mammary fat pad (Hinck & Silberstein, 2005), the number of TEBs in wt and Sharpincpdm mouse mammary gland whole mounts was analysed (Fig 2E). The number of Sharpincpdm TEBs was reduced (Fig 2F), likely reflecting the reduced ductal growth and branching. In histological sections, the overall cellular organisation of the Sharpincpdm TEBs was normal (Fig 2G), and similar numbers of proliferating (BrdU-positive) and apoptotic (cleaved caspase-3-positive) cells were observed in wt and Sharpincpdm mammary gland TEBs (Fig EV2D). Together, these data show that the Sharpincpdm mammary gland has reduced ductal outgrowth in vivo suggesting a previously unknown role for SHARPIN in the regulation of mammary branching morphogenesis. Epithelial SHARPIN is dispensable for mammary gland morphogenesis Since SHARPIN expression was detected in the mature luminal and luminal progenitor cells in the mouse mammary gland, and only at low level in basal epithelial cells (Fig 1D), we postulated that SHARPIN deficiency could affect mammary epithelial regeneration and differentiation. To investigate this, MECs were isolated from pubertal wt and Sharpincpdm mice and surface labelled to detect CD24 expression in combination with CD29 (integrin beta 1) or CD49f (integrin alpha 6) and quantified basal epithelial (LinnegCD24intCD29+ or LinnegCD24intCD49f+) and luminal epithelial (LinnegCD24hiCD29neg or LinnegCD24hiCD49fneg) cell populations (Taddei et al, 2008) (Fig EV2E). Interestingly, both labelling strategies indicated that 50–60% of the MEC population expressed luminal and 30–40% basal markers in both wt and Sharpincpdm samples (Fig EV2E), demonstrating that the Sharpincpdm mammary epithelium has normal proportions of basal and luminal epithelial cells. Next, to conclusively evaluate whether Sharpincpdm mammary epithelium retained normal regenerative capacity, indicative of normal stem cell population, small pieces of wt or Sharpincpdm mammary glands were transplanted into virgin wt recipients' cleared (epithelium-free) mammary fat pads (Fig 3A). Ductal outgrowth from transplants was evaluated from mammary gland whole mounts 7–11 weeks after transplantation. The Sharpincpdm (donor) epithelium was able to fully regenerate the mammary gland in the wt (recipient) stroma (Fig 3A and B) and invade into the fat pad to a similar extent as the wt donor epithelium (Fig 3C). To evaluate the role of SHARPIN in terminal differentiation of the mammary gland, virgin wt mice were transplanted with wt or Sharpincpdm mammary epithelium and mated 10 weeks post-transplantation. Mammary gland whole mounts were prepared 15 days after conception (P15; Fig 3D). In five out of six transplants of both genotypes, mammary epithelium was able to regenerate and undergo tertiary branching and alveologenesis during pregnancy, when grown in wt mammary stroma (Figs 3D and EV2F), demonstrating that epithelial SHARPIN is dispensable for terminal differentiation of the mammary gland. Due to the reduced lifespan of the Sharpincpdm mice (Potter et al, 2014), the reciprocal transplantation of wt epithelium into Sharpincpdm recipients was not feasible. Together, our data demonstrate that wt fat pad stroma is able to support mammary ductal growth and branching, as well as the terminal differentiation of the Sharpincpdm epithelium, suggesting that SHARPIN deficiency is predominantly affecting the mammary stromal compartment. Figure 3. Stromal SHARPIN regulates mammary gland ductal outgrowth during puberty A–C. Monitoring of mammary gland development following transplantation of wt or Sharpincpdm mouse mammary epithelium in the mammary fat pads (epithelium-free) of virgin wt mice. (A) Representative carmine alum-stained images of mouse mammary glands generated from wt or Sharpincpdm epithelial tissue transplants. (B) Quantification of transplant growth take-on-rate (n = 17). (C) Quantification of fat pad filling rate of grown transplants (n = 7–10) in wt hosts. D. Monitoring of mammary gland differentiation during pregnancy following transplantation of wt or Sharpincpdm mouse mammary epithelium into wt hosts. Host animals were mated and mammary glands isolated at P15. Representative carmine alum-stained mammary gland whole mounts generated from wt and Sharpincpdm mammary epithelial transplants (upper panel), and magnifications of the branched ductal epithelium (lower panel) are shown (n = 5 mice). Scale bars represent 1 mm. E. Representative images of carmine alum-stained mouse mammary gland whole mounts generated from 7-week-old S100a4-Cre; Sharpinfl/fl conditional knockout mice and their littermate controls (S1004-Cre; Sharpinfl/+). F. Quantification of the normalised Sharpincpdm and S1004-Cre; Sharpinfl/fl mammary ductal outgrowth area relative to littermate control mice (wt and Sharpincpdm n = 10 glands; S100a4-Cre; Sharpinfl/+ and S100a4-Cre; Sharpinfl/fl n = 8 glands). G. Quantification of the number of TEBs per gland (n = 8–9 glands). Data information: (F, G) Mean ± SEM; Mann–Whitney test. Download figure Download PowerPoint SHARPIN in stromal cells regulates ductal outgrowth in mammary glands The stroma of the developing mammary gland is composed of several different cell types (including fibroblasts, endothelial cells and leukocytes), which undergo complex dialog with the invading mammary epithelium (Reed & Schwertfeger, 2010; Howard & Lu, 2014). Macrophages, eosinop
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