581

Regulation of differentiation by TGF-P

Harold L Moses* and Rosa Serrat

Recent experiments in neural, skeletal, endothelial, and type I receptors are complex and mediate the wide variety
hematopoietic tissues have provided new insights into the of responses of cells to specific TGF-P ligands.
way members of the transforming growth factor-b (TGF-P)
superfamily regulate cellular differentiation. TGF-fi.s regulate Table 1
the fate of multipotential stem cells instructively (in the
Transforming growth factor-0 superfamily*.
neural crest) by regulating the expression or function of
tissue-specific transcription factors, as well as selectively TGF-Ps MIS/lnhibins BMP-related

(in the mesenchyme) by regulating the expression of TGF-!31 MIS BMP-2

required growth factors and their receptors. During skeletal TGF-P2 GDNF BMP-3
development, TGF-fis have unique functions and act TGF-P3 n-inhibin BMP-4
Inhibin-PA BMP-5
sequentially to modulate chondrocyte and osteoblast
Inhibin-pB BMP-G/Vgr-1
differentiation. Responsiveness to TGFj3.s changes as cells Inhibin-PC BMP-7
differentiate and evidence now suggests that changes in GDF-9 BMP-8/OP-2
TGF-P receptor profile may account for some of these GDF-1
GDF-3
differences. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Drosophila and transgenic mouse models are
Vgr-2
now providing useful insights into mechanisms of TGF-P
Nodal
action in ~4~0. DPP
60A
Screw
Addresses
*Partial listing of TGF-P superfamily members from mammals and
*Vanderbilt Cancer Center and Department of Cell Biology,
Drosophila. Those identified only in Xenopus and chicken have been
649 Medical Research Building II, Nashville, Tennesse 37232-6838,
omitted. Adapted from [211. MIS, Miillerian inhibiting substance.
USA; e-mail: hal.moses@mcmail.vanderbilt.edu
tDepartment of Cell Biology, Vanderbilt University Medical Center,
649 Medical Research Building II, Nashville, Tennessee 37232-6838,
USA; e-mail: rosa.serra@mcmail.vanderbilt.edu In this article, we review recent advances in the under-
Current Opinion in Genetics & Development 1996, 6:581-586 standing of the role and mechanism of function of the
TGF-Ps and their receptors in differentiation of selected
0 Current Biology Ltd ISSN 0959-437X
cell types and tissues.
Abbreviations
BMP bone morphogenetic protein
CDMP-1 cartilage-derived morphogenetic protein-l
Neural tissue
dw decapentaplegic Clues to the mechanisms of differentiation in mammalian
GDF growth and differentiation factor tissues can often be inferred from genetic analysis
mbHLH myogenic basic helix-loop-helix of invertebrates. Several signals-including those from
NCSCs neural crest stem cells
members of the TGF-P superfamily-are required for
RA retinoic acid
SF steel factor patterning and differentiation in the eye imaginal disc of
shs shortsighted DrosopMa melanogaster.

The DrosopMa gene decapentaplegic (dpp) encodes a

Introduction member of the TGF-P su p er f amily that was first identified
hlembers of the transforming growth factor-p (TGF-P) as a signal involved in embryonic dorsal/ventral patterning
superfamily are multifunctional peptides that regulate (reviewed in [6]). Certain mutant alleles of dpp also display
cell proliferation, differentiation, and adhesion (Table 1; a small eye phenotype as a result of altered photoreceptor
reviewed in [l,Z]). TGF-Pl, TGF-P2, and TGF-P3 cause development [7]. Differentiation across the eye disc
arrest in the G1 phase of the cell cycle in many proceeds in the posterior to anterior direction. The front of
non-transformed cell types in vitro; they also stimulate differentiation is marked by the morphogenetic furrow, an
matrix production by mesenchymal cells. Most cells indentation that coincides with a stripe of Dpp expression.
secrete TGF-j3s as large latent proteins that require Undifferentiated cells in the eye disc respond to Dpp by
activation-most likely by proteolysis - before becoming first arresting in the G1 phase of the cell cycle and finally
functional. TGF-Ps act through heteromeric complexes differentiating into photoreceptors.
of type I and type II serine/threonine kinases (Table 2;
reviewed in [3-S]). A family of TGF-P serine/threonine A different DrosopMa gene, shortsighted (s/Is), is homolo-
kinase receptor genes have been cloned recently. Type II gous to a mammalian TGF-P responsive gene, TSC-22
receptors can associate with several different type I [8”,9]. There is good genetic evidence that Shs acts
proteins; these interactions between various type II and in the dpp pathway during photoreceptor differentiation;
582 Differentiation and gene regulation

first, _& expression is dependent on Dpp; second, Figure 1 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH

mutations in s/is act synergistically to dpp mutants, and
third, overexpression of .r/ls can partially rescue the eye
phenotype caused by specific dpp mutations [8”]. Shs
and TSC-22 are cytoplasmic leucine zipper proteins and
may act to regulate the DNA-binding activity of specific
transcription factors that ultimately regulate photoreceptor
differentiation. These studies provide a relevant model for
biochemical and molecular studies of the differentiation of
specialized nerve cells in mammals.

Table 2

TGF-P receptor family*.

Type I serine/threonine Ligands Organism 0 1996 C urre nt O p ~m o nin G e ne tic s & De ve lo p m e nt

kinases
ALK-5, TPR-I, R4, RPK-2 TGF-Ps Mammals
Regulation of differentiation in neural tissue by members of the TGF-8
ALK-4, ActR-IB, R-2, SKR-2 Activins Mammals
superfamily. Neural crest cells have the potential to differentiate
ALK-3, BRK-1 BMP-2, BMP-4, BMP-7 Mammals
into several cell lineages including neurons, Schwann cells, and
ALK-6, RPK-1 BMP-2, BMP-4, BMP-7 Mammals
smooth muscle cells. Members of the TGF-8 superfamily bias lineage
ALK-2, Rl, SKR-1, Tsk-7L Activins, TGF-Ps Mammals
decisions by these stem cells. BMP-2 instructs neural crest stem
ALK-1, R3, TSR-l Activins, TGF-Ps Mammals
cells to commit to the neural cell fate. BMP-2 may act by inducing the
Atr-1 Activins Drosophila
expression of a lineage-specific transcription factor, MASH-l. TGF-!31
Thick veins DPP Drosophila
instructs neural crest stem cells to differentiate into smooth muscle
Saxophone DPP Drosophila
and neuregulin results in differentiation to Schwann cells [l 1’4. Once
Daf- 1 7 C. elegans
neural crest cells are committed to the Schwann cell lineage, TGF-fll
promotes the non-myelinating phenotype [17*,18*].
Type II serine/threonine
kinases
ActR-IIA Activins Mammals
ActR-IIB Activins Mammals
TPR-II TGF+s Mammals Later in murine development, and in the adult, TGF-P
Cl4 MIS Mammals
has been shown to regulate further Schwann cell differen-
T-ALK BMP-2, BMP-4, BMP-7 Mammals
Atr-II Activins Drosophila tiation (Fig. 1). Schwann cells can differentiate along two
Punt DPP Drosophila pathways leading to the myelinating or non-myelinating
Daf4 ? C. elegans phenotype (reviewed in [l&13]). Isoforms of TGF-P
*Adapted from [3,4]. MIS, Mtillerian inhibiting substance. have been localized to adult and embryonic central and
peripheral neural tissue [14,1.5]. After injury, neurons
express and release TGF-Ps into the local environment
Members of the TGF-P superfamily have been shown to [13,16]. In co-cultures of neurons and Schwann cells,
promote alternate neural crest cell fates in mouse neural TGF-Pl expression is downregulated by axon/Schwann
crest stem cells (NCSCs) grown in vitro ([ 10,l l”]; Fig. 1). cell interaction [17’]. TGF-P treatment results in the
Bone morphogenetic protein-Z (BMP-2)-a member of proliferation of isolated Schwann cells whereas axon-
the TGF-P superfamily- rapidly induces expression of induced proliferation of Schwann cells in co-culture is
the neural-specific basic helix-loop-helix protein, the inhibited [17-l. Addition of TGF-Pl to purified Schwann
mammalian Achaete-Scute homolog MASH-l, in NCSC cell cultures and co-cultures results in the promotion of
cultures [l lo*]. Up to 75% of NCSC colonies grown in the non-myelinating phenotype [ 17’,18*]. The expression
the presence of BMP-2 were found to be positive for of TGF-P in response to nerve injury and the effects
the neuron-specific marker peripherin by the fourth day on TGF-P on Schwann cell cultures suggest a model
of culture relative to control cultures which contained no in which TGF-P plays an important role zyxwvutsrqponmlkji
in nerve
neurons; in addition, MASH-l expression was localized regeneration [ 17’,18’, 191. These studies also suggest that
near sites of BMP-2 expression in vivo. Taken together, downregulation of TGF-P, as a result of axon/Schwann
these data suggest that BMP-2 regulates MASH-l expres- cell interactions during development and repair following
sion and neurogenesis directly [ 1 lo*]. In contrast, TGF-@l injury, would allow proliferation and differentiation of
promotes smooth muscle differentiation in almost all Schwann cells to progress.
NCSC colonies [l l**]. Neuregulin promotes the formation
of Schwann cells from NCSC cultures [lo]. TGF-P, Skeletal tissue
BMP, and neuregulin are known to act instructively to Members of the TGF-P superfamily appear to act
bias lineage decisions by multipotent NCSCs rather than sequentially in regulating specific aspects of endochondral
by selectively promoting the survival of lineage-specific bone development (reviewed in [20-Z]). BbIP-2, TGF-P
precursors. and BMP-6 are thought to act in a srepwise manner
 Regulation of differentiation by TGF-P Moses and Serra 583

during osteoblast differentiation. BhIP-2 promotes os- more differentiated. A model in which the ratio of
teoblast differentiation in undifferentiated mesenchymal type I to type II receptors regulates responsiveness to
cells [23**,24-261 and alters TGF-P receptor expression, TGF-j3 has been proposed. BMP-2 increases osteoblast
permitting increased osteoblast function in response to differentiation along this panel of cells and alters the
TGF-P [23**]. BhIP-6 and retinoic acid (RA) treatment cell surface expression of TGF-P receptors towards the
have been shown to enhance osteoblastic differentiation of more differentiated pattern, decreased type II receptor
the ROB-C26 pluripotent mesenchymal cell line [24,27’]. expression [23”]. TGF-P was a less effective mitogen
Extracellular matrix from RA-treated or BhlP-6 overex- but was more effective in stimulating collagen synthesis
pressing cells demonstrated zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
in vivo osteoinductive activity and alkaline phosphatase activity in BhIP-2 treated cells
[27*]; this activity was blocked by BhIP-6 antibodies. [23**]. BhIP-2 may act to specify cell surface expression
TGF-@ antibodies inhibited the osteoinductive activity of TGF-P receptors, providing a mechanism for regulation
of extracellular matrix from RA-treated but not BRIP-6 of TGF-P responsiveness and the stepwise regulation of
overexpressing cells. TGF-P expression is induced early skeletal development by TGF-P superfamily members.
in RA-treated cells whereas BRIP-6 expression peaks
later [ZS]. hlembers of the TGF-P superfamily may also hlyoblast differentiation in culture is negatively regulated
act sequentially to regulate chondrocyte differentiation. by TGF-P. hlyogenic basic helix-loop-helix (mbHLH)
TGF-Pl, TGF-P2 and BhIP-4 act synergistically to proteins are muscle-specific transcription factors that were
promote chondrogenesis in chick limb bud mesenchymal first identified by their ability to induce the myogenic
cells [ZS]. TGF-P3 effectively promotes differentiation of phenotype in fibroblasts (reviewed in [37]). TGF-P was
pre-condensation mesenchyme whereas BRIP-2 is most shown to act in a pathway independent of activated
effective in promoting further differentiation of cells that RX p2lval [38] and to suppress the transcriptional
have already begun to differentiate [26]. activity of mbHLH proteins without affecting DNA
binding activity [39]. hlyoblast differentiation occurs only
In addition, the unique functions of each factor in skeletal during a specific time within the G1 phase of the cell
development are demonstrated in mice and humans with cycle. Several pieces of data suggest that components
mutations in genes of the TGF-P superfamily. Rlutations of the cell cycle machinery are linked to myoblast
in Bmp-5 result in the mouse shotlear phenotype [29] and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
differentiation. First, mbHLH proteins associate with
Cdf-5(growth and a’t$+$erentiation _factor- 5) mutations have the retinoblastoma protein (pRb), a known regulator of
been shown to be responsible for brachypodism in mice progression through G1 phase [40,41]. Second, mbHLH
[30]. h,Iutations in the human cartilage- derivedmo@ ogenetic proteins do not promote muscle-specific gene expression
protein- l (CLLM P- 1) gene have been associated with in cells which express defective retinoblastoma protein
Hunter-Thompson type chondrodysplasia [31”]: this is [42]. Third, treatment with TGF-P stimulates proliferation
the first human disorder attributable to a mutation in a and upregulates cyclin Dl expression in myoblasts [43*].
TGF-P superfamily gene. Fourth, overexpression of cyclin Dl prevents myoblast
differentiation [43’,44]. Surprisingly, cyclin D3 expression
The response of cells to TGF-P depends on their was shown recently to be upregulated in the later
differentiation status. When grown to high density, TGF-/3 stages of myoblast differentiation it1 vitro and TGF-P
induces early chondrogenesis in cultures of the mouse prevents the accumulation of cyclin D3 [43’]. It is not
multipotent mesenchymal cell line C3H/lOT1/2 [32]. known if overexpression of cyclin D3 blocks inhibition of
Type II collagen is expressed in response to TGF-P but differentiation by TGF-P.
cells do not attain the typical chondrocyte morphology,
maintaining the appearance of condensed precartilage Blood and vasculature
mesenchyme, suggesting a requirement for additional Bone marrow stromal cells have the potential to dif-
factors [32]. TGF-/3 promotes chondroblast differentiation ferentiate into several mesenchymal lineages, including
of rat calvaria cells plated on matrigel [33]. TGF-P, osteogenic, adipocytic, and hematopoietic fates (reviewed
however, inhibits the formation of hypertrophic cartilage in [45]). TGF-P acts selectively to regulate the fate
and bone mineralization in cultured mouse long bone of these multipotential cells. Steel factor (SF) and its
rudiments [34]; other researchers have also found that receptor, c- kit, are required for the growth of hematopoietic
TGF-j3 blocks the conversion of mature chondrocytes to precursors. TGF-P inhibits the transcription of SF in
hypertrophic cells [35,36]. The regulation of osteoblast bone marrow stroma and accelerates the degradation
differentiation by TGFj3.s is also dependent on the of c- kit in hematopoietic precursors, thereby prevent-
differentiation status of the cells. Alkaline phosphatase ing hematopoiesis [46]. In apparent contrast to results
activity is inhibited by TGF-P in early osteoblasts but is obtained for adult hematopoiesis, 50% of homozygous
stimulated in the highly differentiated ROS 17/2.8 cell line TGF-Pl null mice are known to have defects in yolk
[23”]. The profile of cell surface TGF-j3 receptors was sac hematopoiesis and vasculogenesis [47**]. It is possible
shown to differ along this osteoblast lineage [23**]-the that distinct molecular controls for embryonic and adult
level of type II and type III receptors diminished but hematopoiesis exist; it is known, for example, that
type I receptor expression was maintained as cells became embryonic hematopoiesis is independent of SF and c- kit
584 Differentiation and gene regulation

[48]. Unfortunately, the TGF-Pl null embryos that show tumor suppressor gene, DPC4 [55*]. The function of
defective yolk sac hematopoiesis and vasculogenesis die these proteins in the TGF-P regulation of differentiation
before the onset of hepatic hematopoiesis, therefore this remains to be elucidated.
hypothesis could not be tested.
Differentiation-specific promoters can be used to over-
TGF-P inhibits the growth of endothelial cells grown express TGF-Ps in transgenic mice. This strategy could
in standard cell culture [49], however, no endothelial provide information on how TGF-P affects cells in their
hyperplasia was detected in TGF-Pl null embryos [47**]. natural environment, with normal extracellular matrix and
This observation suggests that the primary direct effect cellular contacts. In a recent study, overexpression of
of TGF-Pl in viva is not on growth but on the control TGF-Pl in distal lung epithelium of 16.5 and 18.5 days
of cellular differentiation. The vascular phenotype of the post co&m mouse embryos resulted recently in the
TGF-Pl null mice was similar to that seen in fibronectin inhibition of epithelial differentiation as measured by
null animals [SO]; it is possible, therefore, that TGF-Pl Clara cell secretory protein and pro-surfactant protein C
regulates the endothelial phenotype by controlling the expression [56*]; abnormal expression of type I collagen
expression of fibronectin and thus regulating adhesion. In and smooth muscle actin may inhibit expansion of
addition, growth of endothelial cells in three-dimensional distal airways, resulting in defective sacculation and
culture is not inhibited by TGF-Pl but differentiation differentiation. In another study [57*], overexpression of
of capillary tube-like structures is promoted [Sl”]. Cells TGF-P2 late in osteoblast differentiation results in an
in three-dimensional culture demonstrate a higher ratio osteoporosis-like phenotype. This result was unexpected,
of type I to type II receptors relative to cells grown in as TGF-P2 increases osteoblast function in culture and
two-dimensional culture [Sl”]. TGF-P responsiveness is exogenously administered TGF-P2 results in increased
dependent on cellular adhesion as well as differentiation bone matrix in vivo. The authors suggest that TGFj32,
status: alterations in the TGF-P receptor profile appear to when deposited into the bone matrix, increases the rate of
mediate these differences. bone remodeling by affecting molecular mechanisms that
coordinate osteoblast and osteoclast activity. These studies
Conclusions underscore the complexity of cell, extracellular matrix, and
At present, several themes regarding the regulation of growth factor interactions and demonstrate the importance
differentiation by TGF-P are emerging. First, TGF-Ps can of in vlejo experimentation.
regulate the fates of multipotent cells either instructively
or selectively [11**,46]. Second, members of the TGF-P Loss of function mutants can be used to address the
superfamily regulate differentiation sequentially role of endogenous
along growth factors in differentiation in
specific lineages [23**,29,30]. Third, responsiveness vivo. Dominant-negative
to the receptor mutations act to inhibit
TGF-Ps is dependent on the adhesion and differentiation endogenous receptor function by competing for ligand,
status of the cell and alterations in TGF-P receptor substrate, or other accessory proteins. Truncated, kinase-
profile may account at least in part for the differences deficient type II TGF-P receptors-which bind ligand
in responsiveness [23”,51”]. Many issues still remain and heteromerize with endogenous receptors-have been
concerning the mechanisms by which TGF-Ps shown to block responsiveness
regu- of cells in vitro to isoforms
late differentiation. Genetic and molecular data suggest of TGF-P [58-611. Depending on the promoter used,
that the TGF-Ps may regulate differentiation-specific responsiveness to TGF-P could be blocked in specific
transcription directly [8”,11”,38]. TGF-P may also regu- tissues at specific developmental stages in transgenic
late differentiation indirectly by influencing cell adhesion animals. This strategy reduces the problems associated
[29,32,47**,52]. Finally, regulation of differentiation by with embryonic lethality and functional redundancy in
TGF-P may be linked to regulation of proliferation in genetically null animals and, additionally, the response to
specific circumstances [43’,46]. all TGF-P isoforms is blocked. Preliminary data from our
laboratory demonstrate that overexpression of a dominant-
Genetic studies in zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Drosophila have been useful in negative TGF-P type II receptor in mammary gland
outlining evolutionarily conserved pathways of TGF-P epithelium or in cartilage results in inappropriate cellular
signaling. Genetic analysis has implicated a role for S/is in differentiation (HL Moses eta/, unpublished data). These
Dpp-mediated regulation of photoreceptor differentiation. data suggest that signaling by TGF-Ps through the
TSC-22, a mammalian cytoplasmic leucine zipper protein, type II receptor is required for the maintenance of normal
is homologous to Shs and may also be involved in mammary gland and chondrocyte differentiation.
regulation of differentiation by TGF-j3 [8”]. Several
additional genes involved in Dpp signaling have been Little is known about the mechanism of TGF-P-
identified recently and are known to have homology to mediated differentiation in TJ~VO.Clues from experiments
mammalian genes. The Drosophila schnuti gene encodes in invertebrates and cell culture can provide a starting
a protein homologous to the mammalian transcription point for further biochemical and molecular experi-
factor PRDII-BFl [53’,54*] and the Drosophila Mothers mentation in V~VO. Cross-breeding of specific transgenic
againstdpp (Mad) genes have homology to a novel human and knockout mice could provide a genetic model for
 Regulation of differentiation by TGF-P Moses and Serra 585

TGF-P signaling pathways. Eventually, a combination 15. Unsicker K, Flanders KC, Cissel DS, Lafyatis R, Sporn MB:
Transforming growth factor-p isoforms in the adult rat
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B, Munaut C, Rigo JM, Lefebvre PP, Octave J-N et a/.:
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