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Regulation of Mammary Morphogenesis: Evidence for Extracellular Matrix-Mediated Inhibition of Ductal Budding by Transforming Growth Factor-PI GARY B. SILBERSTEIN,*~'KATHLEENC. FLAmERs,t ANITAB.ROBERTS,-~ AND CHARLESW.DANIEL*

Branching morphogenesis in the mammary gland involves focal regions of cell proliferation, the terminal and lateral ductal buds, that exist simultaneously with extensive regions of differentiated ducts in which budding and growth are actively suppressed. Exogenous transforming growth factor-/j1 (TGF-61) has previously heen shown to locally inhibit the formation and growth of mammary ductal buds. Here we report that endogenous TGF-/jl, produced by epithelial and stromal mammary cells, forms complexes with extracellular matrix (ECM) molecules surrounding those ductal structures in which budding is inhibited. The largest amounts of immunostainahle TGF-$1 are found in mature periductal ECM, and the least in newly synthesized ECM. In all areas of active ductal growth, where DNA-synthetic buds were forming new ductal branches, we found a highly focal loss of TGF-/jl from the periductal ECM at the hud-forming region of the duct. When growth of the new buds terminated, the structures again became associated with TGF-$-rich ECM. These findings indicate that ECM must reach a certain state of maturity before it becomes associated with TGF-/jl and that TGF-/31 can be depleted selectively from the periductal ECM at focal growth points. A different type of growth point, the alveolar (secretory) buds, was also investigated. These buds are known not to be inhihited by exogenous TGF+l, and we found them not to be associated with changes in ECM-hound TGF-01. Our results support the concept that the periductal ECM acts as a reservoir for TGF-/31 that functions to maintain an open pattern of mammary t 1992 Academic Press, Inc. branching hy inhibiting ductal, hut not alveolar, hud formation. INTRODUCTION

Transforming growth factor-p1 (TGF-pl), the best studied member of the TGF-fl family of growth factors, is thought to act by autocrine or paracrine mechanisms to modulate cell division and extracellular matrix synthesis/deposition (see Roberts and Sporn, 1989; Rizzino, 1988 for reviews). Both TGF-fll and its receptor are found in most cells and tissues, but the receptors are not obviously modulated and are therefore poor candidates for altering TGF-@l activity (Roberts and Sporn, 1989; Massague et al., 1990). This has raised questions concerning the mechanisms by which TGF$l action is regulated and localized in developing tissues. TGF-pl is secreted in a latent form, leading to the hypothesis that local activation could be a means of controlling activity (Roberts and Sporn, 1989; Lyons et al., 1990). The recent discovery of extracellular matrix elements with specific affinities for TGF-01 has raised the possibility of a second regulatory mechanism, compatible with the site-specific activation hypothesis, in which TGF-/jl is sequestered within the extracellular matrix near its target. In this model, the extracellular matrix is

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viewed as a reservoir that can accumulate as well as release TGF-I31 (see Ruoslahti and Yamaguchi, 1991 for a review). The small proteoglycan decorin, for example, inhibits TGF-Pl action by mediating formation of a reversible complex between TGF$l and Type I collagen (Yamaguchi et al., 1990; Yamaguchi and Ruoslahti, 1988). It could therefore modulate TGF-01 activity by binding it to collagen for later release. Our recent investigation of endogenous TGF-0 isoforms in the mouse mammary gland demonstrated that TGF-@1 and $3 messages and protein are expressed in glands from young, mature, and pregnant, but not lactating, animals (Robinson et ul., 1991). Considering that extracellular matrix-associated TGF-@l may be an important regulatory mechanism, it was noteworthy that TGF-pl was detected in high concentrations in the collagenous extracellular matrix immediately adjacent to growth-inhibited ducts. In a comparable study, the embryonic lung synthesized a TGF-pl-rich, collagenous matrix around developing alveoli coincident with morphogenetic stabilization (Heine et al., 1990). Other studies from our laboratory indicate a role for TGF-01 in ductal morphogenesis. When implanted by means of slow-release implants into gland containing actively growing ducts, TGF-El had two major effects. First, within 12 hr epithelial (but not stromal) DNA 354

synthesis in ductal end buds was inhibited. This inhibition was reversible and, by several criteria, mimicked normal growth regulation (Daniel et (xl., 1989). Second, epithelium-dependent synthesis of a collagenous extracellular matrix was rapidly induced (Silberstein ef al., 1990). As in the developing lung, morphogenetic stabilization of the developing mammary gland was coordinated with the epithelium-dependent induction of a collagenous ductal matrix (Silberstein and Daniel, 1982, 1984). Different results were obtained when TGF-pl was introduced into mammary glands in early pregnancy, when alveolar rather than ductal buds were forming. Growth of alveolar buds was not inhibited by TGF-01, and ectopic ECM synthesis was not observed. If it is hypothesized that an extracellular form of TGF-/jl participates in growth autoregulation, then growth-related changes in the extracellular localization of TGF-/jl might be predicted, based on a model in which the ECM acts as a TGF-$1 reservoir. Two hypotheses were tested. First, we examined the possibility that extracellular matrix surrounding the mammary duct acts as a true reservoir that can acquire as well as lose TGF-$1. Second, should there be changes in TGF-01 levels in the matrix, these would be coordinated with wellcharacterized aspects of ductal morphogenesis. Here we report that the periductal ECM is a TGF-01 reservoir in which the levels of TGF-81 immunoreactivity change in a manner consistent with a role for TGF-$1 in regulating ductal branching. MATERIALS AND METIIOI)S d~lin/oIs. C57/BL/crl mice were used for all studies. TissztcJ l)r~jpcxt.rrt iota .fbr~ iw ~nll?,olocrxlixcxfiorr. Fixation was accomplished by perfusion (Kiernan, 1981). Briefly, animals were heparinized (SC injection; 1 U/g body wt) up to 2 hr before perfusion, anesthetized, and, after puncturing the left ventricle with a blunted hypodermic needle, perfused with 1% and then 4%’ freshly prepared paraformaldehyde (30 and 60 ml, respectively). After perfusion, tissue was left i/r siflr for 30 min, excised, postfixed in Bouin’s solution for an additional 5 hr, transferred to ‘70%) ethanol, embedded in paraffin, and sectioned at 5 pm. hfihorlic~s. The mature domain of TGF+l was detected using anti-50-75, prepared against a peptide comprising the correspondingly numbered amino acids. In the mammary gland, anti-50-75 detected primarily extracellular protein. On Western blots anti-50-75 does not cross-react with TGF-p2; TGF-133 was not tested. (K. Flanders, unpublished). The amino-terminal portion of the pro-region was detected with anti-266-278 (Flanders cuttrl.. 1989; Wakcfield et trl., 1988). The antibodies were

used as IgG fractions prepared by protein A-Sepharose chromatography (Flanders et d., 1988). Sfcxinixg protocol. TGF-61 was localized in sections essentially as described by Heine et trl. (1987), using an avidin-biotin-peroxidase kit (Vector Laboratories Inc., Burlingame, CA). Deparaffinized sections were first treated to several blocking steps (30 min each; 0.2% glytine to block endogenous aldehydes; 0.3% hydrogen peroxide/methanol to block endogenous peroxidase; and 5% goat serum) and then incubated with IgG fractions of antibody (ea. 15 &ml) overnight at room temperature. After incubation, sections were washed in 1% goat serum followed by the detection kit protocol. Sections were lightly counterstained with hematoxylin. Sfuiniug spec$kify. Specificity for TGF-/jl peptide epitopes was determined for anti-266-278 by incubating tissue sections with primary antibody that had been preincubated 2 hr at room temperature with a 20-fold molar excess of the cognate peptide. For anti-50-75, antiserum was passed over a column of Sepharose resin conjugated with peptide 50-75 to remove specific antibodies (Heine et ol., 1987). For routine controls, the primary antibody was replaced with protein A-purified, normal rabbit IgG which resulted in ablation of staining. Iderpretufio?r 0.i’ in1 ~7mosfain i?t.q resuIts. The esistence of a latent form of TGF-fil complicates the interpretation of immunolocalization results. For this study, intracellular localization (in similar structures, not in the same section) of the pro-region was judged to define a site of TGF-bl synthesis. While extracellular (or intracellular) stain for only the mature domains possibly indicates the presence of active TGF-/jl. Implar~ts. EVAc (Elvax 40P) was a gift of DuPont Chemical Co. (Ilniversal City, CA). Implant preparation is described in detail elsewhere (Silberstein and Daniel, 1987). RESIJLTS

The typical pattern of extracellular TGF-01 staining around a mature duct is illustrated in Fig. 1A. Heavy TGF-01 immunoreactivity was seen in the periductal ECM as well as in unformed collagenous elements in the fatty stroma. Stain was associated with fibers in both cases. Specificity of the antibody for TGF-PI was demonstrated by greatly reduced staining with antiserum extracted with TGF-Bl peptide-conjugated Sepharose (Fig. 1B). A TGF-fil reservoir might be filled rapidly or relatively slowly. In the latter case, it would be expected that newly formed matrix would be negative for TGF-/31 immunoreactivity, which would then be acquired pro-

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illustrating the accumulation of TGF-01 in the mammary extracellular matrix. (A) Mature duct in gland from FIG. 1. Photomicrographs 5-week-old animal. Stain was concentrated in the compact periductal stroma (large arrow) and was also seen in fibrous stroma not associated with epithelium (small arrow) and around adipocytes (Bar = 10 pm). (B) Specificity of staining for TGF-Pl. A section adjacent to A was incubated with antibody that had been treated with Sepharose conjugated to the cognate peptide (Bar = 10 pm). (C) End bud (large arrow) with two lateral buds (small arrows). Staining in the periductal matrix was absent around the duct immediately subtending the end bud and light beneath the lateral buds (open triangle), becoming heavier lower on the duct. The duct departed the plane of the section at the solid triangle (Bar = 7.5 pm). (D) Duct subtending a two-lobed end bud. The two lobes of the end bud are partially out of the frame on the upper left. A gradient of staining in the periductal stroma can be seen in the continuous fibrous sheath along the duct. Staining was very light around the flank of the end bud (small solid arrow) and increased in intensity further down the duct to levels seen around mature ducts (large solid arrow). The duct from which the end buds grew runs horizontal to the base of the photograph and out of the plane of the section (double-headed arrow). A collagenous septae also stained heavily (open arrow). Where this septae meets the compact stroma around the end bud (open triangle), its deep stain contrasts with the very light stain in the newly formed matrix. Where the septae meets the matrix around the mature duct, staining blends with periductal matrix (solid triangle). (Bar 2 10 pm).

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of Ductul Budding

by TGF-@I

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FIG. 2. Photomicrographs illustrating the extracellular localization of TGF-@l during ductal lateral branch initiation (A,B), elongation (C,D), termination (E), and alveolar budding (F). (A) Incipient lateral bud from a mature duct. A thick layer of ECM is still present over the bud tip (open arrow), but staining was absent when compared with the densely staining periductal ECM (solid arrow points to ductal epithelial cells). The unstained zone was approximately the same thickness as the heavily stained matrix over the flanking, morphogenetically quiescent duct (Bar z 13 bm). (B) Lateral bud, slightly further grown out compared to that in A. Stain was absent over the bud tip (open arrolr) and was diminished on the Ranks (large solid arrows) compared with staining of the contiguous matrix over the subjacent duct. Again, loss of stain could not be attributed to obvious loss of ECM, u-hich was still present around the tip. Staining over the mature duct appeared heaviest in the matrix immcdiatelp adjacent to myoepithelial cells (small arrows) (Bar = 20 lm). (C) A lateral branch has breached the heavily stained, compact, periductal matrix (large arrows). In contrast to earlier stages of bud formation, little ECM is seen on the branch, and the tip appears to he in direct contact with surrounding adipocytes. Stain also appears on the basal surface of the base of the branch (small arrows) (Bar = 17 pm). (D) Extended lateral branch. Stained, fibrous stroma extends onto the newly formed branch (arrows). Note stained collagenous septae (triangle) (Bar 7 10 pm). (E) Lateral branch with continuous, TGF-pl-rich sheath. A dense, thin, TGF-@l-rich sheath surrounded this lateral branch and appears to be continuous with a similar structure on the main duct (open triangles), where it is overlaid by a looser, thick fibrous sheath (solid arrolv). Synthesis of a thickened fibrous sheath on the branch is also visible (solid triangles) (Bar = 20 pm). (F) Alveolar hud (solid arrow) intiuccd by an intragland implant of DCA. The bud is approximately the same size as ductal buds shown in A and is superficially similar, except that immunostaining for TGF-$1 in the overlying ECM is not reduced (open arrow) (Bar = 13 pm).

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FIG. :;. PhotomicroKr~lr)tls illustrating the localization of an epitope to the precursor domain of TGF-rll in morphogeneticallg inactive and xtivcs ductal tissue. (A) Terminal tlurt. Staining was obscrvcd in most of the ductal epithclial cells (large arrow), as well as in some stromal cells (open arrow). .2lywpithelinl cells appc~ar~~l not to stain (small arrows). Note that the sheath of highly cellular. compact stroma xvas unstained (curver! ;wow) (Bar ‘LOpm). (B) Slwiiicity 01’staining for TGF-$1. A section adjacent to A xas incubated with an antibody preparation that had hwn previously treated n-it h a Wf’old molar wcess of the cognate peptide (amino acids 266-27X in the TGF-dl precursor domain). Staining \vas compl~~tc~ly al,l;ltecI t F;:\r 16 pm 1. ((‘1 End bud. Most cells in the body of the end bud Lvere immunopositive. The monolayer of myoepithelial stem wlls that vstcnds ;rrounti thr bud did not stain (curved arrows). Note that the leading edge of the end bud (from the point of maximum diameter, forward I is frcv of compact, fibrous stroma. The end bud flank is heavily invested in fibrous connective tissue that ~vas negative for the prcvzursc~r c>pitopc (larger arrowI (Rar Iti pm). (I)) Incipient lateral branch off of mature duct. TGF-$1.positive tiuctal cells were prcwnt in tht, lateral hurl tip (largcs arrow) :md as \v(~II ax in the atljawnt, morphoggcnetically quiescent ductal cpithelium. This bud has breached the compxi. lwritluctal strom;l, which is unstainc~tl tcurvcd arrows). Many atiilxwytes \vvcr(’ TGF-dl-positive (small arrows) (Bar 20 pm 1.

gressively as the tissue matured. This possibility was investigated by comparing TGF-01 staining in newly formed ductal ECM immediately behind end buds with mature ECM found along developmentally stabilized ducts. An end bud, its newly formed duct, and two lateral branches are shown in Fig. 1C. No TGF-pl immunoreactivitg was seen immediately behind the large terminal end bud or in association with the two smaller lateral buds. TGF-@1 had accumulated in the stroma behind the two lateral branches, but not in the newer fibrous sheath between the end bud and the branches. Staining became heavier with increasing distance from the end bud; in this type of branching morphogenesis, distance is equivalent to developmental maturation. Details of staining in a newly formed sheath are shown in Fig. 1D. Adjacent to the end bud staining was light, while further down the duct staining intensity increased to a level typically seen on mature, growth-static structures. Collagenous septae (Fig. 2D) provide structural support for the fatty stroma and develop prior to and independent of the ductal epithelium. In contrast to the gradient of TGF$l stain around the end bud and subtending duct, stain on a nearby septae spanning the same zone as the duct was uniform and heavy along its entire length. This provided a natural, positive control for the adjacent light TGF-PI staining around the epithelium.

Lateral branches can occasionally arise from mature ducts that are encased in a thick, TGF-@l-rich matrix, as well as from newly formed ducts a short distance behind an end bud. We searched stained mammary gland preparations for occasional adventitious ductal buds, which were removed for either immunostaining or thymidine autoradiography. Small buds were always associated with substantially increased levels of DNA synthesis, which continued until the growth in the branch was terminated (Fig. 4B). This growth termination was always accompanied by the formation of ECM over the ductal tip (Fig. ZE). Thus, the presence of a cap of organized ECM proved to be a reliable indicator of growth termination, and conversely the absence of fibrous tissue around the bud was tightly correlated with elevated levels of DNA synthesis. Along mature ducts, incipient branches first appear as small, rounded elevations in the ductal epithelial sheet and its overlying ECM; elongation proceeds when the bud breaches the collagenous sheath and extends into the fatty stroma (Fig. 2). If extracellular matrixassociated TGF$l plays an inhibitory role, changes in its localization might be expected in these highly local-

ized zones of transition from growth-quiescent ducts to growing buds. An incipient branch (bud stage) is shown in Fig. 2A. Staining for extracellular TGF-fll was lost in the matrix over the bud. The transition from stained to unstained matrix was abrupt and coincided with the base of’ the bud. Figure 2B illustrates a slightly more elongated bud. Deep immunostaining of the ECM fibers stopped abruptly at the base of the bud, although a few lightly stained fibers appear to extend around the tip. The heaviest immunostain was immediately adjacent to the myoepithelial cells that extend longitudinally in a monolayer over the ductal epithelium. Loss of immunoreactivity over incipient buds could not be ascribed to prior loss of ECM in the region. Figure 2A and 2B show that a layer of unstained ECM, approximately equal in thickness to the deeply staining periductal ECM, is still present at this stage, indicating that loss of immunostaining preceded complete loss of ECM. Figure 2C shows a ductal branch in a more advanced stage of elongation, in which little or no observable matrix sheath is present, and the nascent branch tip appears to be in direct contact with surrounding adipocytes. Having breached the periductal matrix a lateral bud, like a terminal end bud, induces a new, TGF-/jl-rich collagenous sheath around its subtending duct. This process is illustrated in Figs. 2C-2E. The new breach was clearly defined in Fig. 2C, where strands of stained fibers run parallel to the axis of the main duct only to the neck of the new branch. On the neck of the branch, a few lines of stain at the epithelial-stromal interface indicate newly acquired TGF$l. A longer branch (Fig. 2D) showed TGF-PI-stained matrix extending approximately halfway up its length. In Fig. 2E, a lateral branch is illustrated that has reestablished a TGF-Blrich ECM sheath along its entire length and around the tip. This pattern is characteristic of branches which have terminated growth due to the presence of adjacent mammary ducts (not shown).

The cellular origin of the TGF-01 observed in the periductal ECM was investigated by immunostaining for an epitope of the precursor domain of pro-TGF-/jl. This has been correlated with ilr sift hybridizations for TGF01 mRNA (Robinson et ul., 1991) and can be considered evidence for a site of synthesis. Pro-TGF-@1 was identified in the majority of mammary ductal cells whether the structure was growing (Figs. 3C and RD) or not (Figs. 3A and 3B). In contrast, cells within mature as well as newly formed periductal matrix did not stain. Myoepithelial cells were mostly unstained (Fig. 311) as

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FIG. 4. (A) Induction of alveolar budding hy introduction into the mammary gland of a slow-release plastic implant containing 150 pg DCA (large arrow). The mouse was a 3-month-old harmonally intact female. Small arrows, alveolar buds that are most numerous close to the implant. (Bar = 100 pm). (B) DNA synthesis in cells of a ductal bud. The &week-old mouse vas injected with (3Hlthymidine 30 min before the mammary glands were removed for autoradiographg. Arrows indicate labeled cells (Bar = (50 Km).

were the cap cells, their stem cell precursors on t,he end bud (Fig. 3C). Alveolar

Buds

The early development of secretory alveoli appears superficially similar to ductal budding, but represents an alternate developmental pathway that is not inhibited by exogenous TGF-Pl (Daniel et al., 1989). Both pregnant mice and mice that had received deoxycorticosterone acetate (DCA) implants into the mammary gland were used. DCA implants were useful in stimulating a gradient of alveolar budding, in which buds at different stages of development could be readily located (Fig. 4A). Results were identical whether the alveoli buds were induced by DCA or pregnancy; ECM over alveolar buds did not show the loss of immunoreactivity seen in ductal buds of comparable size (Fig. ZF). DISCUSSION

Numerous in vitro studies have convincingly established that various extracellular matrix molecules can

form complexes with growth factors (reviewed in Ruoslahti and Yamaguchi, 1991). From these studies the concept has emerged that the extracellular matrix may normally serve as a reservoir for growth factors, modulating their action by selective, focal accumulation and release. This idea is attractive since growth factors are predicted to act over short distances by autocrine or paracrine circuits, and the extracellular matrix may create highly localized microenvironments that are either enriched or depleted of important cell regulatory factors. By definition, a true reservoir (as opposed to a trap or sink) is dynamic, its contents demonstrably in flux; to date we are aware of no previous evidence for the operation of such a reservoir in a developing system. While TGF-fll is demonstrably present in the extracellular matrix of mouse embryonic and adult, tissue (Flanders ef ~1..1989; Thompson et al., 1989; Heine et al., 1987), including the mammary gland (Fig. 1A and Robinson ef al., 1991), little understanding for TGF-01 dynamics can be gleaned from these observations. In the present study, the conclusion that periductal ECM TGF-@l was acquired, maintained, and lost during ductal growth and morphogenesis constitutes strong evidence that this matrix is a true TGF-/LO reservoir.

SILBERSTEINETAL.

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Inhibition qf’I?uctal Budding bg TGF-@I

Acquisition and Loss qf TGF-fll in the Periductal ECM The induction of a periductal fibrous sheath immediately behind the end bud and on the neck of lateral branches (Fig. 1) is coordinated with ductal morphogenesis. TGF-Bl appeared well after the end bud sheath had formed, in zones that had been previously shown to be rich in Type I collagen and other extracellular matrix macromolecules (Silberstein and Daniel, 1982; Williams and Daniel, 1983; Silberstein et a,l., 1990). The staining was graded in intensity, with the lightest stainingoccurring in relatively newly synthesized matrix and the heaviest in the more mature matrix. Since the staining gradient did not reflect obvious differences in TGF-Pl synthesis by epithelium or stroma proximal to the periductal ECM (Fig. 3), it seems reasonable to suggest that the gradient reflect,s the dynamics of TGF-/31 acquisition. Periductal ECM-associated TGF-fll was not apparently synthesized by fibrocytes within the matrix, since these did not stain for intracellular pro-TGF$l (Fig. 3), and must therefore have come from the epithelium or from other stromal cells, such as adipocytes, that were positive for pro-TGF-fil. A stromal contribution appears possible because of staining in collagenous septae (Figs. lA, 1D and 2E). The emergence of a lateral branch from a growthquiescent duct reflects precisely localized relief from growth inhibition, followed by focal disruption of the periductal ECM. It is clear that TGF-Pl immunoreactivity in the periductal ECM was lost very early in the budding process. Absence of staining was dramatic well before the growing branch had breached the matrix which, in the earliest stages, was still the same thickness over the tip of the bud as over the flanking duct (Fig. 2A). The focal loss of TGF-fll immunoreactivity in the periductal ECM could reflect release of bound TGF/?l or, alternatively, steric changes that make TGF-01 invisible to the antibody. In either case the morphological picture is consistent with changes in TGF-Pl that may result in altered availability or biological activity. The acquisition and maintenance of TGF-61 in the periductal ECM (Fig. 1) is consistent with the hypothesis that specific macromolecules within these structures immobilize TGF-/31. The basal lamina contains Type IV collagen and integrin (unpublished observation), which can bind the RGD sequence of the TGF-fll latency protein (Roberts and Sporn, 1989). In addition to collagen I, the periductal ECM contains fibronectin, which binds but does not inactivate TGF-/31 (Bissell and Hall, 198’7; Fava and McClure, 1987). The synthesis of the aforementioned molecules is stimulated by TGF-@l, suggesting that by a feedback circuit TGF-/31 could modulate the formation of its own reservoir.

The Regulation

?f Mammary Ductal Branching

The inhibition of mammary ductal branching is an active process that is required for the maintenance of pattern. Ductal epithelium is not terminally differentiated (transplanted fragments will quickly grow a new ductal system if transplanted into available mammary adipose tissue) and yet, the open spaces between ducts are maintained under hormonal conditions that are optimal for rapid ductal budding and growth (Faulkin and DeOme, 1960). A specific role for ECM-associated TGF-/X in chronic inhibition of lateral budding is inferred from several observations. Exogenous, implanted TGF-@l reversibly inhibited ductal growth and budding, and ducts did not become refractory to prolonged exposure (Daniel et al., 1989). TGF-/31 was acquired in the periductal ECM during the transition from active growth to a growth-static state. The apparent loss of TGF-61 from the ECM occurred only over ductal buds. Alveolar buds are not affected by TGF-01, nor did we observe changes in TGF-bl associated with the ECM. A focal loss of periductal TGF-Pl would simultaneously accomplish two things crucial to ductal budding. First, ductal epithelium would be free to proliferate in response to ambient mitogens. Second, the matrix-stimulating effects of TGF-Pl, which include a multifaceted inhibition of matrix degrading proteinases, would be lost, allowing degradation of the periductal ECM. Breaching of the fibrous collagen over a new branch indicates the highly localized action of matrix collagenases and would be consistent with focal relief of enzyme inhibition. The regulatory and biochemical mechanisms underlying the focal loss of TGF$l are of interest, in understanding proliferative and metastatic aspects of human mammary neoplasias, of which greater than 90% are ductal in origin. If the growth-static status of ducts is normally maintained by a flux of TGF$l through the ECM reservoir, possible changes in this autoregulatory mechanism may lead to changes in growth control. Also, since metastatic ductal neoplasms must breach the basal lamina and periductal ECM, events similar to those described here for normal branching may also have a role in the ontogeny of metastasis. We thank Phyllis Strickland and Kathy nical assistance. This work was supported

VanHorn for skillful techby PHS Grant HD 2’7845.

REFERENCES Bissell, M. J., and Hall, H. G. (1987). Form and function in the mammary gland: The role of extracellular matrix. h “The Mammary Gland: Development, Regulation, and Function,” (M. C. Neville, and C. W. Daniel, Eds.), pp. 97-146. Plenum, New York. Daniel, C. W., Silberstein, G. B., Van Horn, K., Strickland, P., and

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Robinson, S. (1989). TGF-beta-l-induced inhibition of mouse mammary ductal growth: Developmental specificity and characterization. Del-. Rio/. 135, 20-30. Faulkin, L. J., Jr., and DeOme, K. B. (1989). Regulation of growth and spacing of gland elements in the mammary fat pad of the C3H mouse. J. 1Z;clt/.Ctrwe~ I~st. 24, 953-968. Fava, R. A., and McClure, D. B. (19X7). Fibronectin-associated transforming growth factor. .1 Cell. Physiol. 131, 184-189. Flanders, K. C., Roherts, A. B., Ling, N., Fleurdelys, B. E., and Sporn, hf. B. (1988). Antibodies to peptide determinants in transforming growth factor beta and their applications. Biochcwis~r.!/ 27,7X-746. Flanders, K. C., Thompson, N. L., Cissel, D. S., Van Obherghen-Schilling, E., Baker, C. C., Kass, M. E., Roberts, A. B., Ellingsworth,L. R., and Sporn, M. B. (1989). Transforming growth factor beta-l: Histochemical localization with antibodies to different epitopes. J. W/ Bid. 108, 653-660. Heine, IT. I., Munoz, E. F., Flanders, K. C., Ellingsworth, L. R., Lam, P. H., Thompson, N. L., Roberts, A. B., and Sporn, M. B. (1987). Role of transforming growth factor heta in the development of the mouse embryo. J. &I/ Biol. 105, 2861-28’76. Heine, U. I., Munoz, E. F., Flanders, K. C., Roberts, A. B., and Sporn, M. B. (1990). Colocalization of TGF-beta-l and collagen I and III, fibronectin and glycosaminoglgcans during lung branching mor109, 29-K phogenesis. Dew/opwu/f Kiernan, J. A. (1981). “Histological and Histochemical Methods: Theory and Practice,” pp. 23-24. Pergamon Press, New York. Lyons, R. M., Gentry, L. E., Purchio, A. F., and Moses, H. L. (1990). Mechanism of activation of latent recombinant transforming growth factor beta-l by plasmin. J. Cc// Biol. 110,1361-1367. Massague, J., Cheifetz, S., Boyd, F. T., and Andres, J. L. (1990). TGFbeta receptors and TGF-beta binding proteoglgcans: Recent progress in identifying their functional properties. A,(r,. XI;: Acrtrl. SC;. 593,59-72. Rizzino, A. (198X). Transforming growth factor beta: Multiple effects on cell differentiation and extracellular matrices. Dcr: Bid. 130,

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Roherts, A. B., and Sporn, M. B. (1989). The transforming growth factor-betas. 1~ “Peptide Growth Factors and Their Receptors” (M. B. Sporn, and A. B. Roherts, Eds.). Springer-Verlag, Heidelberg. Robinson, S. D., Silbcrstein, G. B., Roberts, A. B., Flanders, K. C., and Daniel, C. W. (7991). Regulated expression and growth inhibitory effects of transforming growth factor-beta isoforms in mouse mammary gland development. 11f~w/ol))r/(~tlt 113, 867-878. Ruoslahti, E., and Yamaguchi, Y. (1991). Proteoglgcans as modulators of growth factor activities. (‘(>/I 64, 867-869. Silberstein, G. B., and Daniel, C. R. (1982). Glycosaminoglycans in the basal lamina and extracellular matrix of the developing mouse mammary duct. L)ct%.Biol. 90, 215-222. Silberstein, G. Il., and Daniel, C. W. (1984). Glycosaminoglycans in the basal lamina and the extracellular matrix of serially aged mouse mammary ducts. dZwl/. .lgc,. DC>/,.24, 151-162. Silberstein, G. B., and Daniel, C. 1%‘.(1987). Investigation of mouse mammary ductal growth regulation using slow-release plastic implants. ?J.I1triry Sci 70, 1981l1990. Silberstein, G. B., Daniel, C. W., Coleman, S., and Strickland, P. (1990). Epithelium-dependent induction of mouse mammary gland extracellular matrix t)?; TGF-beta-l. X Cdl Kid. 110, 2209-2219. Thompson, N. I,., Flanders, K. C., Smith, M., Ellingsworth, L. R., Roherts, A. B., and Sporn, M. B. (1989). Expression of transforming growth factor beta-l in specific cells and tissues of adult and neonatal mice. .I, (‘c/I Biol. 108, 661-669. Wakeheld, I,. M., Smith, D. M., Flanders, K. C., and Sporn, 111.8. (1988). Latent transforming growth factor-beta from human platelets. ./. Bio/. Urew. 263, 76467654. Williams, J. M., and Daniel, C. W. (1983). Mammary ductal elongation: Differentiation of myocpithelium during branching morphogenesis. DPP. Bid. 97, 274-290. Yamaguchi, Y., and Ruoslahti, E. (1988). Expression of human proteoglgcan in chincsc hamster ovary cells inhibits cell proliferation. Ec/f,rw 336 ‘124-246. Yamaguchi, G.yMann, I). M., and Ruoslahti, E. (1990). Negative regulation of transforming growth factor-beta by the proteoglgcan decorin. ,Yrcrfitw 346, 281-X84.

Regulation of mammary morphogenesis: evidence for extracellular matrix-mediated inhibition of ductal budding by transforming growth factor-beta 1.

Branching morphogenesis in the mammary gland involves focal regions of cell proliferation, the terminal and lateral ductal buds, that exist simultaneo...
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