Ltbp1Lis focally induced in embryonic mammary mesenchyme, demarcates the ductal luminal lineage and is upregulated during involution
Latent TGFβ binding proteins (LTBPs) govern TGFβ presentation and activation and are important for elastogenesis. Although TGFβ is well-known as a tumor suppressor and metastasis promoter, and LTBP1 is elevated in two distinct breast cancer metastasis signatures, LTBPs have not been studied in the normal mammary gland.
To address this we have examined Ltbp1 promoter activity throughout mammary development using an Ltbp1L-LacZ reporter as well as expression of both Ltbp1L and 1S mRNA and protein by qRT-PCR, immunofluorescence and flow cytometry.
Our data show that Ltbp1L is transcribed coincident with lumen formation, providing a rare marker distinguishing ductal from alveolar luminal lineages. Ltbp1L and Ltbp1S are silent during lactation but robustly induced during involution, peaking at the stage when the remodeling process becomes irreversible. Ltbp1L is also induced within the embryonic mammary mesenchyme and maintained within nipple smooth muscle cells and myofibroblasts. Ltbp1 protein exclusively ensheaths ducts and side branches.
These data show Ltbp1 is transcriptionally regulated in a dynamic manner that is likely to impose significant spatial restriction on TGFβ bioavailability during mammary development. We hypothesize that Ltbp1 functions in a mechanosensory capacity to establish and maintain ductal luminal cell fate, support and detect ductal distension, trigger irreversible involution, and facilitate nipple sphincter function.
KeywordsMammary Gland Elastic Fiber Mammary Development Luminal Cell Luminal Progenitor
Bacterial artificial chromosome
Bone morphogenetic protein
Cluster of differentiation
Epidermal growth factor
Epithelial to mesenchymal transition
Lymphoid enhancer-binding factor 1
Large latent complex
Latent TGFβ binding protein
Dihydrocyclopyrroloindole tripeptide minor groove binder
Nuclear fast red
Pregnancy-associated breast cancer
Phosphate buffered saline
Quantitative reverse transcriptase-polymerase chain reaction
Rough endoplasmic reticulum
Stem cell antigen 1
Small latent complex
Smooth muscle actin
Terminal end bud
Transforming growth factor β
Transforming growth factor β Receptor
Latent transforming growth factor β (TGFβ) binding proteins (LTBPs) are regulators of elastogenesis and TGFβ . Their critical role in tissue development, homeostasis and resilience is demonstrated by the fact that LTBP loss-of-function mutations underpin a growing list of human genetic syndromes [2, 3, 4]. Gain of LTBP gene expression also has pathological consequences: LTBP1 is upregulated in two breast cancer metastasis signatures and is one of only six genes found in common to both [5, 6].
Ltbp genes encode a family of secreted proteins, Ltbp1-4, that show extensive sequence homology to fibrillins, which polymerize to form microfibrils and coat elastic fibers [1, 7]. Ltbp proteins are initially deposited onto fibronectin and later transferred to microfibrils by interaction with fibrillins . Their importance for the structural integrity and tensile function of the extracellular matrix (ECM) is illustrated by the pathologies seen in Ltbp4S-null mice resulting from defective elastic-fiber formation in the intestine, lung and pulmonary artery and in humans with Urban-Rifkin-Davis syndrome [4, 9, 10].
In addition to their contribution to ECM structure, Ltbp1, Ltbp3 and to a lesser extent Ltbp4 govern the spatial patterning and activation of TGFβ. TGFβs are secreted in a latent form, encapsulated by their cleaved latency-associated propeptide (LAP), and deposited within the ECM for subsequent activation. Ltbps post-translationally regulate TGFβ in three ways. First, they chaperone the association of TGFβ with LAP and through preferential binding affinities control which of three TGFβ isoforms emerge from the cell . Second, Ltbps incorporate latent TGFβ within the ECM thereby determining where TGFβ is presented to its receptors . Third, Ltbps provide a key link between the ECM and the cell surface that is essential for stretch activation of TGFβ [13, 14, 15]. Both integrins and Ltbp bind to LAP. Thus, when Ltbp1 is anchored in a stiff ECM and stress fibers exert tension on integrins, conformational changes occur in LAP that lead to release of the active TGFβ [13, 14, 16]. One major response to TGFβ signaling is synthesis of new matrix proteins . Thus, Ltbps create a mechanosensory system that generates a highly localized feedback response to cell traction or tension within the microenvironment [1, 18].
Mouse mutants have illuminated the roles of Ltbps in tissue homeostasis and their involvement in human pathology. Ltbp1 hypomorphs show facial dysmorphia  and Ltbp1L loss leads to embryonic lethality due to heart malformation , Ltbp2 loss-of-function mutations cause glaucoma in humans and lens defects in mice , Ltbp3 loss-of-function mutation results in severe bone malformation [3, 22, 23] and Ltbp4S-null mice show multiple organ defects [4, 9, 10]. In some mutants the prevailing pathology reflects compromised elastogenesis [10, 24]. In others the phenotype can be ameliorated by concurrent deletion or pharmacological antagonism of TGFβ, supporting the central role of Ltbps in TGFβ biology and pathology .
Three TGFβ isoforms are differentially expressed and exert multiple effects during mammary development . Loss- and gain-of-function studies have shown that TGFβ signaling restrains pubertal ductal extension and side branching by stimulating Wnt5a expression [26, 27, 28, 29, 30, 31]. TGFβ1 influences stem cell regenerative potency and cell-fate determination and has been proposed to suppress precocious alveologenesis in the adult gland prior to pregnancy [27, 32, 33, 34, 35, 36]. Weaning massively induces TGFβ3 expression, and this surge is essential for the demise of the differentiated glandular epithelium and remodeling events during mammary involution [37, 38]. TGFβ1 has also been the object of intense investigation due to its pathological relevance for breast cancer [39, 40] where it acts as a tumor suppressor in premalignant lesions and at later stages promotes metastasis through induction of epithelial-to-mesenchymal transition (EMT).
Knowledge of Ltbp's temporal and spatial expression pattern is central to understanding TGFβ signaling both in the physiological setting of the normal mammary gland and in breast cancer. Yet to date there have been no studies on Ltbp within the normal mammary gland. Here we show that Ltbp1 is induced in a highly specific temporal and spatial pattern throughout mammary development, supporting the concept that dynamic transcriptional regulation of Ltbp1 provides a mechanism to impose considerable restriction on TGFβ bioavailability. Ltbp1L is upregulated early during embryonic mammary mesenchyme specification and is sustained in smooth muscles of the nipple sphincter. Within the mammary gland, Ltbp1L is induced exclusively in the ductal luminal epithelium but is silent in alveoli and therefore provides a rare biomarker distinguishing ductal from alveolar luminal lineages. Ltbp1 protein is deposited around basal cells of all ducts and side branches, and lies in close proximity to elastic fibers that exclusively encase the permanent ductal system. Ltbp1 is prominently upregulated during involution, with kinetics similar to that reported for TGFβ3, suggesting important functions in gland remodeling.
Carmine staining of mammary whole mounts revealed no differences between Ltbp1L lz/+ mice and wild-type littermates in ductal elongation, branching, alveolar development or involution. Pups from both genotypes faired equally well in terms of weight gain (data not shown). We concluded that Ltbp1L lz/+ mice show no evidence of haploinsufficiency and justified their use to study the regulated expression of Ltbp1L during mammary development. Staging of pregnancy and embryos were performed by daily checking of vaginal plugs, with noon of the day of the plug considered day 0.5. Embryonic stages were confirmed by determining the degree of limb development as indicated in Theiler’s classification of mouse development (The Atlas of Mouse Development, MH Kaufman).
Mice and embryos were screened by 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) staining of tails and confirmed by PCR analysis. Genomic DNA was prepared from 0.5 cm of tail by digesting overnight in 0.5 ml digestion buffer (50 mM Tris–HCl pH 7.4, 100 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 0.5% SDS, 200 μg/ml proteinase K). Then 150 μl of 5 M NaCl was added and the digest was agitated for 15 minutes on a rotator: 500 μl of supernatant was collected after centrifugation at 14,000 G for 15 minutes, and subjected to two rounds of ethanol precipitation. The final pellet was resuspended in 200 μl TE (10 mM Tris–HCl pH 7.4, 1 mM EDTA) and 1 μl was added to a 20-μl PCR. Thirty cycles of PCR (94°C, 58°C and 72°C for 1 minute each) were carried out. The wild-type Ltbp1L allele was detected by amplification of a 430-bp band using forward 5′-CTTAGTTCCTCCATCCTTCC-3′ and reverse 5′-CAGACTTCACCTTCCCAGGG-3′ primers. The Ltbp1Llz/+ knock-in allele was detected in a separate reaction using the forward primer listed above and a reverse primer 5-GTCTGTCCTAGCTTCCTCACTG-3′ (see Figure 1B arrowheads) to amplify a 440-bp product. The gender of embryos was determined by amplification of the Sry gene on the Y chromosome (forward primer: 5′-GAGAGCATGGAGGGCCAT-3′ and reverse primer: 5′-CCACTCCTCTGTGACACT-3′). Amplification products were resolved by electrophoresis on 2% agarose gels run for 30 minutes in TAE electrophoresis buffer (40 mM Tris-acetate, 1 mM EDTA).
X-Gal staining of embryos and mammary gland whole mounts
Embryonic day (E) 10.0 to E15.5 embryos were dissected and fixed in 4% paraformaldehyde (PFA) (Sigma Aldrich, St Louis, MO, USA) prepared in PBS for 20 to 50 minutes depending on the stage. Skin with attached mammary fat pads was removed from E16.5 to E18.5 embryos and stretched carefully on cardboard, and mammary glands from adult mice were dissected and flattened onto glass slides then fixed in 4% PFA for 30 minutes. Following fixation, samples were washed 4 × 15 minutes with rinse buffer (2 mM MgCl2, 0.1% sodium deoxycholate, 0.2% NP40 prepared in PBS) and stained in X-Gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal, Denville Scientific, South Plainfield, NJ, USA) prepared in rinse buffer) at room temperature for 2 to 3 h. After staining, samples were rinsed twice in PBS and post-fixed in 4% PFA overnight at 4°C, dehydrated through an ethanol gradient (2 × 10 minutes in 70%, 95%, and 100% ethanol), then placed in Carnoys’s fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) followed by Citrisolv reagent (Fisher Scientific, Pittsburgh, PA, USA) to clear the fat.
Whole-mount carmine staining
X-Gal stained mammary glands were rehydrated in a reverse-graded series of ethanol washed in water and then stained for 1 h in carmine solution diluted 1:5 in water. Carmine was prepared by boiling 1 g carmine alum and 2.5 g aluminium potassium sulphate in 500 ml of water for 20 minutes followed by filtration. The glands were dehydrated in a graded ethanol series, cleared in Carnoy’s solution, placed in Citrisolv for 30 minutes, and mounted in Cytoseal (VWR, Radnor, PA, USA). Glands were then viewed using a Zeiss Axiovert (Oberkochen, FRG) brightfield microscope.
Histology and immunodetection
E10.5-stage embryos were embedded in 10% gelatin, sectioned at 70 μm with a vibratome, and mounted with Fluoromount G (Southern Biotech, Birmingham, AL, USA). Older embryos and mammary glands were processed for X-Gal staining and fixation as described above. Isopropanol was substituted for xylene to prevent diffusion of the X-Gal stain during processing and tissues were embedded in paraffin and sectioned. Sections (4 μm) were placed on Superfrost Plus slides, baked 1 h at 60°C and deparaffinized for 5 minutes in Citrisolv for X-Gal-stained tissues. Tissues were then rehydrated through a reverse gradient of ethanol solutions. For histology, sections were stained with 0.1% solution of Nuclear Fast Red (NFR) (Polyscientific, Bayshore, NY, USA) for 1 minute. Tissues were then dehydrated and dipped in xylene (or Citrisolv in the case of X-Gal-stained tissues) before being mounted in Cytoseal (VWR). For immunohistochemistry (IHC), citric acid antigen retrieval was performed by submerging the slide containing deparaffinized 4-μm sections in 10 mM sodium citrate solution (pH 6.0) and boiling in a microwave at 90% power for 30 minutes, followed by quenching of endogenous peroxidase using 3% hydrogen peroxide. Primary mouse antibodies against smooth-muscle actin (SMA) 1 (1:500, DAKO, Carpinteria, CA, USA), estrogen receptor (1:500, DAKO), p63 (1:1,000 LabVision, Kalamazoo, MI, USA), and rabbit antibodies against Cytokeratin 14 (1:8,000, Covance, Princeton, NJ, USA), Lef-1 (1:100 Cell Signaling, Danvers, MA, USA), androgen receptor (1:500, Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and guinea pig antibodies against Vimentin (1:1,000, Progen) were added overnight at 4°C. For IHC, biotin-labeled secondary antibodies (1:1,000) and streptavidin-horseradish peroxidase (HRP) (1:200, Vector Laboratories, Burlingame, CA, USA) were added for 30 minutes each, and colorimetrically detected with diaminobenzidine (Vector Labs). Frozen 5-μm sections were stained with rabbit antibodies against LTBP (Ab39 , 1:200, a gift from Dr Carl-Henrik Heldin, Uppsala University, Sweden, and rL1C , 1:100, a gift from Dr Lynn Sakai, Portland Shriners Research Center, Portland, OR, USA), tropoelastin (1:500, Elastin Products Company, Inc., Owensville, MO, USA), and mouse anti-SMA, described above, were detected by Cy3-labeled donkey anti-rabbit (Fisher Scientific) and Alexafluor-488-labeled donkey anti-mouse secondary antibodies (Life Technologies Inc, Carlsbad, CA, USA). Bioreagent (4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) from Sigma Aldrich) was used for immunofluorescent localization of nuclei in confocal images. Elastic fibers were also detected by staining with Wiegert’s resorcin-fuchsin for 1 minute .
Mammary epithelial cell preparation and flow cytometry
The third, fourth and fifth mammary glands from 8- to 16-week-old virgins were dissected, inguinal lymph nodes were discarded, and the mammary glands were minced between two scalpels into a fine paste. The tissue was dissociated for 6 h at 37°C in collagenase/hyaluronidase solution (catalog number 07912, Stem Cell Technologies Inc., Vancouver, BC, Canada), and further dissociated with 0.25% Trypsin-EDTA and 10 mg/ml dispase (catalog number 07913, Stem Cell Technologies) with 1 mg/ml DNase, before filtering through a 40-μm mesh. Endothelial and hematopoietic lineages were depleted using antibodies to TER119, CD45, CD140a, and CD31 (1:100, Becton Dickenson (BD), Franklin Lakes, NJ), with three separations on an EasySep magnet. Primary antibodies CD49f-PerCP-Cy5.5 (1:200, BD), CD24-PE (1:400, BD), CD29-Pacific Blue (1:200, Biolegend, San Diego, CA, USA), CD61-APC (1:200, CalTag MedSystems, Buckingham, UK), stem cell antigen 1 (Sca1)-phycoerythrin (PE) (1:400, BD) were added for 30 minutes at 4°C. Fluorescein Di-β-D-Galactopyranoside (FDG-gal) loading was performed after primary antibody staining, according to the manufacturer’s instructions (FluoReporter Kit, Life Technologies, Green Island, NY, USA). Flow cytometry was performed on a BD LSRII or BD FacsCalibur, and analyzed using FlowJo v8.7.
RNA isolation and qRT-PCR analysis
The fourth and fifth pair of mammary glands were harvested from wild-type mice at different stages of postnatal mammary development, dissected and snap-frozen in liquid nitrogen. A block of tissue approximately 0.5 × 0.5 × 0.5 cm was homogenized for 5 minutes in 1 ml of TRI-Reagent (Life Technologies) using a hand-held tissue homogenizer (Kinematica, Lucerne, Switzerland), then mixed with 200 μl of chloroform and centrifuged at 14,000 G for 15 minutes to eliminate protein debris. The upper aqueous phase was mixed with an equal volume of 70% ethanol and passed through a Qiagen RNeasy mini spin column by a brief 15 sec centrifugation at 8,000 G at room temperature. Total RNA bound to the column filters was washed in 350 μl of ethanol-containing buffer (RW1 buffer; Qiagen, Valencia, CA, USA) to remove contaminants and incubated in 10 μl of RNase-free DNase I enzyme (273 Kunitz units; Qiagen) for 15 minutes at room temperature to ensure digestion of any residual genomic DNA fragments. The columns were washed according to the manufacturer’s instructions in ethanol-containing buffers (RW1 and RPE buffers; Qiagen). Total RNA was eluted in 50 μl of RNase-free water, and its concentration was analyzed by Nanodrop measurement. Reverse transcription was performed using 2 μl of RNA (10 ng/μl) from tissue using the QuantiTect Probe RT-PCR Kit (Qiagen; catalog number 204443). Real-time analysis was performed using the Taqman Gene Expression Assay (Applied Biosystems by Life Technologies; catalog number 4331182) for mouse Ltbp1 (Mm00498255_m1), Ltbp1L (Mm01226402_m1 spanning exons 1 and 2), and Ltbp1S (custom assay with forward primer: 5′-TTCCAAGGCAAGTTCATGGATA-3′, within intron 4; reverse primer: 5′-AGGAGTAGAGGCAGACAGAGAAAGA-3′, within the fifth exon of Ltbp1 genomic sequence and MGB probe: 5′-6FAM-TAAGCTGATGTGTTTGTTG-3′-MGBNFQ) and mouse β2-microglobulin (Mm00437762_m1). Real-time analysis was performed in the Applied Biosystems ViiA™ 7. Total Ltbp1, Ltbp1L and 1S mRNA levels were normalized to those of mouse β2-microglobulin and plotted as levels relative to tissue from 12-week-old virgins.
Ltbp1L-LacZ expression underlies a route for axillary cell migration and is an early marker of the mammary mesenchyme
Mesenchymal Ltbp1Lactivity accompanies nipple induction and persists in smooth-muscle cells and myofibroblasts in the adult
Ltbp1Lpromoter activity coincides with ductal lumena formation within the embryonic mammary tree
Ltbp1 mRNA is dynamically modulated during postnatal development
Ltbp1Lis induced in ductal luminal cells and distinguishes them from alveolar lineages
To determine more precisely where the Ltbp1L promoter is activated during postnatal mammary development, we examined Ltbp1L-LacZ expression in whole mounts and histological sections. In pubertal mice a balance of proliferation and apoptosis within outer cap and inner multilayered body cells of the bulbous terminal end buds (TEBs) generates the permanent ductal tree and creates a lumen in the subtending ductal system. X-Gal-stained whole mounts revealed Ltbp1L-LacZ expression lining the lumen of the TEB (Figure 7C). Reporter expression was notably absent from the vast majority of body cells, which are considered to be actively proliferating luminal precursors (Figure 7D).
Ltbp1Lactivity is dramatically upregulated during involution
Ltbp1 and elastin encase the mammary ductal system
The importance of TGFβ signaling for mammary physiology and pathology has been well documented however the factors that regulate TGFβ presentation and activation are less well-understood . Although LTBPs determine the spatial deposition of latent TGFβ and thus define the coordinates for its subsequent activation, surprisingly nothing is known about them in normal mammary gland. Here we show that Ltbp1 is dynamically and focally regulated throughout mammary development. The major findings of our study are that 1) Within the mammary epithelium, Ltbp1L is transcribed exclusively by ductal luminal cells and distinguishes them from the alveolar luminal lineage; 2) Ltbp1 protein and elastic fibers exclusively encase the ductal system; 3) Ltbp1L and 1S are upregulated during involution, a developmental window linked to high risk for breast cancer promotion; and 4) Ltbp1L is induced in mammary mesenchyme and sustained in the smooth-muscle cells of the nipple sphincter.
Ltbp1Lis induced in embryonic mammary mesenchyme and persists in nipple sphincter cells
Ltbp1L-LacZ is first expressed in an arc around the forelimb. This pattern is intriguing in light of reports that mammary precursors destined for placodes 1 to 3 migrate along a similar path . It is well known that TGFβ signaling promotes EMT and motogenesis. Thus, Ltbp1L expression may designate a migratory route and potentially stimulate ectodermal cell migration by presenting a focal source of TGFβ. Ltbp1L is next upregulated in the specialized mammary mesenchyme, which plays a pivotal role in inducing mammary morphogenesis and specifying the embryonic nipple and areola . To date there have been no reports of TGFβ involvement in these inductive processes, although other members of the TGFβ family, such as bone morphogenic protein (BMP)4, are known to play critical roles [54, 55]. We find that the expression of mammary mesenchymal markers remains unperturbed and embryonic mammary development proceeds normally in Ltbp1L lz/lz embryos, indicating that Ltbp1L is not essential for mammary mesenchyme specification or inductive function. These results do not, however, preclude the possibility that the products of Ltbp1S, which is expressed from an independent promoter, or other Ltbp genes may compensate . Alternatively Ltbp1L may function at later stages in the differentiation of these cell types. Ltbp1L-LacZ expression persists within smooth muscle cells aligned in radial arrays under the areola, which facilitate nipple projection and regulate the nipple sphincter during milk let-down. There have been no studies on TGFβ in the nipple, however, misexpression of Wnt5a, a target gene of TGFβ, has been shown to impair milk ejection, supporting the concept that specific levels of TGFβ signaling may be critical for nipple function . We also observe strong Ltbp1L-LacZ expression in myofibroblasts during mid-pregnancy when the stroma synthesizes elastin to provide structural support for the lactiferous duct . Whether Ltbp1L functions to reinforce the surrounding elastic fibers, and/or serves in a mechanosensory capacity between TGFβ signaling and the establishment of the unique nipple stroma, remains to be determined.
Ltbp1 and ductal cell fate
Ltbp1L activity is a consistent marker of the ductal luminal lineage, appearing in the embryo at the first sign of ductal canalization. This specificity is maintained throughout pubertal development and pregnancy where it serves as a rare marker distinguishing ductal from alveolar luminal cells. Transplantation studies have suggested that ductal and alveolar progenitors are distinct, but little is known about differences between mature ductal and alveolar luminal cell-types . Ltbp1L is active in approximately 65% of luminal cells but silent within the inner body cells of the TEB, which are thought to be a proliferative progenitor population. It is upregulated within mature CD61-Sca1+ cells in the subtending duct and within a small subpopulation of CD61+ luminal progenitors, which we speculate may generate side branches during pregnancy. Previous studies have implicated TGFβ signaling in suppressing proliferation of luminal populations and maintaining the potency of basal stem cell populations [34, 36, 60]. Our results show that Ltbp1 protein is deposited in close apposition to basal cells encasing the ductal system and thereby positioning TGFβ to carry out these functions.
Ltbp1 in the physiology of ductal dilation and distension
The appearance of Ltbp1L-LacZ expression coincident with lumen formation in the embryonic mammary rudiment and in the pubertal TEB suggests Ltbp1 may position TGFβ to generate lumen by inducing apoptosis . TGFβ is a well-known pro-apoptotic cytokine and multiple studies have demonstrated a role for apoptotic factors in lumen formation in vitro and in vivo[34, 37]. However the periductal restriction of Ltbp1 protein in close association with elastic fibers makes this function unlikely and moreover indicates that they participate in some ductal versus alveolar specific process. A distinguishing feature of ducts is that their lumen remain open at all times. Whether Ltbp1 serves to physically support the open ducts by reinforcing their elastic fiber encasement and/or positions TGFβ to monitor ductal lumenal diameter in a mechanosensory fashion remains to be determined.
Ltbp1Lis silenced during lactation and dramatically induced during involution
The most dramatic changes in Ltbp1 activity occurred with the onset and cessation of lactation. Ltbp1, 1L and 1S mRNA were undetectable during lactation, and Ltbp1L-LacZ expression was lost even from the ducts as the entire epithelium assumed a secretory phenotype and the lumen became engorged with milk. This loss of Ltbp1L and 1S expression coincides with a change in the trafficking of latent TGFβ from basolateral secretion as a large latent complex destined for incorporation into the ECM in an Ltbp-dependent manner to apical secretion of small latent complex into milk, which functions to promote IgA production and induce oral tolerance in the newborn .
Ltbp1L is dramatically induced during involution. Involution is a biphasic event, marked by distinct biological processes. For up to 48 h after weaning the process is reversible and characterized by alveolar apoptosis. After this point it becomes irreversible, as protease-mediated matrix remodeling leads to alveolar collapse and rebuilding of the ECM, to return the gland to a virgin-like state . Teat-sealing experiments have shown that ductal distension triggers involution even in the presence of circulating lactogenic hormones, highlighting the role of local factors . Our results show that Ltbp1L and 1S are induced within 24 h and peak at day 3 of involution, remaining elevated for some days. This pattern is similar to that reported for TGFβ3 in several microarray studies [64, 65]. TGFβ3 is upregulated 6-fold within 3 h of weaning and has been implicated as a local factor triggering alveolar apoptosis, however, the mechanism for its activation has not been studied . Whether Ltbp1 is expressed early enough to facilitate TGFβ3’s role in apoptosis remains to be determined. The peak of Ltbp1 and TGFβ3 induction correlates with the transition to the irreversible stage of involution, suggesting that elevated TGFβ signaling may contribute to this transition. Little is known about the role of TGFβ3 in later involution, though it has been hypothesized to promote fibroblast migration and ECM generation based on the upregulation of wound healing and ECM genes that are targets of TGFβ signaling during this phase [17, 63, 65]. Alternatively, the localization of Ltbp1 protein along ducts suggests it may function to protect the permanent ductal system and its ductal stem cells from destruction by integrating integrin and TGFβ signaling, which promote cell survival and stem cell potency, respectively . Lastly, our finding that Ltbp1 expression is dramatically elevated during involution, when taken collectively with the fact that LTBP1 appears in two metastatic signatures [5, 6] and regulates TGFβ, a factor inducing EMT, suggests that LTBP1 may be a prometastatic element in pregnancy-associated breast cancer (PABC). Detected postpartum, PABC is highly aggressive and this feature is thought to result from the action of prometastatic factors in the microenvironment of involuting glands . Thus LTBP1 levels may be worthy of investigation as a risk factor.
In conclusion, our results establish that Ltbp1 is dynamically regulated during mammary development. The pattern of Ltbp1L activity and Ltbp1 protein localization suggest roles in reinforcing elastic support and mechanosensory feedback for mammary ducts and nipple. Currently nothing is known of the role of this important TGFβ regulator in human breast. Its elevation during involution suggests LTBP1 is worthy of further investigation as a prometastatic candidate in PABC.
We thank Drs Daniel Rifkin (NYUSOM), Carl-Henrik Heldin (Uppsala University, Sweden) and Lynn Sakai (Portland Shriners Research Center, Portland, OR) for LTBP reagents. Our colleagues John Munger, Matthias Kugler and Mary Helen Barcellos-Hoff provided valuable comments on the manuscript. We also thank Peter Lopez of the NYU Cancer Institute Flow Cytometry and Cell Sorting Center, Drs Ram Dasgupta and Sujash Chatterjee for their expertise and help with confocal imaging, Alejo Mujica for the design of a TaqMan qRT-PCR assay specific for the short form of the Ltbp1 mRNA and Lakeisha Esau, Lisa Koetz, Nikhil Sharma and Cassandra Williams provided technical assistance with experiments. This work was supported by DOD-BC123572 and NIH-CA129905 awarded to PC, postdoctoral fellowship DOD-BC112418 awarded to AC, NIH-T32-GM66704-8 training fellowship to JS and NIH training fellowship F31CA130137 awarded to AP. Additionally this work was supported in part by Grant 5P30CA016087-33 from NCI (Flow Cytometry Core New York University (NYU) Cancer Institute). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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