Activins Are Critical Modulators of Growth and Survival
Chester W. Brown,
Liunan Li,
Dianne E. Houston-Hawkins and
Martin M. Matzuk
Departments of Molecular and Human Genetics (C.W.B., L.L., D.E.H.-H., M.M.M.), Pediatrics (C.W.B.), Pathology (M.M.M.), and Molecular and Cellular Biology (M.M.M.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Chester W. Brown, M.D., Ph.D., Assistant Professor, Departments of Molecular and Human Genetics and Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: cbrown{at}bcm.tmc.edu.
 |
ABSTRACT
|
---|
Activins ßA and ßB (encoded by Inhba and Inhbb genes, respectively) are related members of the TGF-ß superfamily. Previously, we generated mice with an Inhba knock-in allele (InhbaBK) that directs the expression of activin ßB protein in the spatiotemporal pattern of activin ßA. These mice were small and had shortened life spans, both influenced by the dose of the hypomorphic InhbaBK allele. To understand the mechanism(s) underlying these abnormalities, we now examine growth plates, liver, and kidney and analyze IGF-I, GH, and major urinary proteins. Our studies show that activins modulate the biological effects of IGF-I without substantial effects on GH, and that activin signaling deficiency also has modest effects on hepatic and renal function. To assess the relative influences of activin ßA and activin ßB, we produced mice that express activin ßB from the InhbaBK allele, and not from its endogenous Inhbb locus. InhbaBK/BK, Inhbb-/- mice have failure of eyelid fusion at birth and demonstrate more severe effects on somatic growth and survival than either of the corresponding single homozygous mutants, showing that somatic growth and life span are supported by both activins ßA and ßB, although activin ßA plays a more substantial role.
 |
INTRODUCTION
|
---|
THE TGF-ß SUPERFAMILY is comprised of a structurally similar, though functionally diverse group of proteins that play important roles in embryonic development as well as the functions of terminally differentiated tissues (1, 2). Activins are members of the TGF-ß superfamily that participate in several biological processes that include the synthesis and secretion of FSH (3), pancreatic islet function (4), response to vascular injury (5), erythropoiesis (6), and inflammation (7, 8) in addition to their extensively studied direct roles in the ovary and testis (9, 10).
Activins ßA and ßB (encoded by Inhba and Inhbb genes, respectively) are closely related, sharing 63% identity and 87% similarity in their mature regions at the amino acid level (11). These proteins associate to form activin A or B homodimers (ßA: ßA or ßB: ßB) or activin AB heterodimers (ßA: ßB). We have shown that mice with a homozygous null mutation at the Inhba locus (Inhba-/-) fail to suckle, have disruption of whisker, palate, and tooth development, and die shortly after birth (12, 13, 14). In contrast, Inhbb homozygous null mutant mice (Inhbb-/-) have eyelid closure defects, a modest prolongation of the gestational period, and an inability to nurse their young. However, Inhbb-/- mice are viable and fertile (15, 16). The differences in the severity of the activin ßA and ßB null phenotypes and results from our laboratory (17), suggest that the different spatiotemporal expression patterns for the activin subunits in vivo are responsible for their unique biological functions.
To better understand the compensatory potential of functionally related proteins in early development, we produced mice with a knock-in of activin ßB into the Inhba locus. The resulting knock-in allele (Inhbatm2Zuk, hereafter, InhbaBK) produces a hybrid activin ßA: ßB precursor protein that is processed to a biologically active activin ßB dimer at sites where activin ßA is normally expressed. This was demonstrated by in situ hybridization during embryonic development (17). In addition, we have shown that the levels of activin ßB protein produced from the knock-in allele are comparable to the levels of wild-type activin ßA protein by examining activin ßA and ßB protein production from ovaries of mutant and control mice (17). Ovaries from mice that were heterozygous for the knock-in allele and also homozygous for the activin ßB null allele (InhbaBK/+, Inhbb-/-) produced activin ßB and activin ßA proteins at comparable levels (17). Although the craniofacial phenotypes and neonatal lethality resulting from the Inhba-/- genotype were rescued in the knock-in mice, additional phenotypes were observed including postnatal growth delay and increased mortality, both findings influenced by the dose of the knock-in allele. These observations suggested that despite similar activities in vitro and some overlapping functions in vivo, activins ßA and ßB are not functionally interchangeable in all settings, and that the InhbaBK allele is functionally hypomorphic relative to the wild-type Inhba allele.
For this paper, we sought to understand the mechanism(s) that contribute to the growth deficiencies and decreased survival of InhbaBK mice, with emphasis on the roles of GH, IGF-I, and hepatic and renal function. In addition, we assess the contribution of endogenous activin ßB signaling to these biological processes by studying double mutant mice that express activin ßB from the Inhba locus, and also have a reduced or absent expression of activin ßB from the Inhbb locus.
 |
RESULTS
|
---|
Growth Plate Analysis of Activin ßB Knock-In Mice Reveals Morphologic Abnormalities
To determine the mechanisms that lead to the growth deficiencies of InhbaBK mice, we examined the proximal tibial growth plates of 3-wk-old wild-type, InhbaBK/BK and InhbaBK/- mice (Fig. 1
, AI). Three-week-old mice were chosen for these and subsequent analyses because InhbaBK/- mice do not survive beyond 4 wk (17). Grossly, the dissected tibiae and other bones of InhbaBK/BK and InhbaBK/- mice were proportionately smaller than those of wild-type mice (not shown). The measured growth plate thickness and the thickness of the proliferative and hypertrophic zones were significantly different for all experimental groups, showing a decrease from wild-type to InhbaBK/BK to InhbaBK/- mice (Fig. 1J
). The same phenomenon was observed for the cross sectional area of cells within the hypertrophic zone (Fig. 1J
). In addition, many cells within the hypertrophic zones of InhbaBK/- growth plates were relatively devoid of cytoplasmic material and the nuclei of those cells lacked the characteristic apoptotic appearance that normally occurs as the cells of the hypertrophic zone transition and become mineralized (Fig. 1
, GI).

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 1. Growth Plate Histology and Quantitative Analysis
AC, Low power view of proximal tibial growth plates. Note the greater width of the proximal tibia and greater thickness of the growth plate (GP) in the wild-type (WT) mouse (A) relative to the same region of tibias from InhbaBK/BK and InhbaBK/- mice shown at the same magnification. DF, The germinal zones (GZ) of WT, InhbaBK/BK and InhbaBK/- growth plates are similar in organization; however, both the proliferative (PZ) and hypertrophic (HZ) zones of InhbaBK/BK and InhbaBK/- mice are underdeveloped, with the hypertrophic zone most affected. GI, Higher power view of cells from the hypertrophic zone showing the greater cell volume and glycogen content (arrowheads) of cells from wild-type mice (G). Note the poorly delineated nuclei undergoing the apoptotic changes that are characteristic for this zone and the absence of these changes in the cells shown from the same zone from InhbaBK/- mice (I). These cells with intact nuclei and relatively pale cytoplasm were occasionally observed, though at a much lower frequency, in InhbaBK/BK mice, and only rarely in wild-type mice. J, Measurements of growth plates from wild-type, InhbaBK/BK, and InhbaBK/- mice. Measurements of the total growth plate thickness (GP), the proliferative zone (PZ), and hypertrophic zone (HZ), as well as the area of cells within the hypertrophic zone, were all significantly different when the groups were compared (asterisks), with the greatest differences observed in the hypertrophic zone. P < 0.0001.
|
|
IGF-I Levels Are Reduced in Activin ßB Knock-In Mice
The abnormalities of the InhbaBK/- growth plates are similar to those reported previously for IGF-I (Igf1) knockout mice. Igf1-/- mice have prenatal and postnatal growth deficiencies, which have been hypothesized to be due in part to the absence of the nutritive insulin-like effects of IGF-I on cells of the hypertrophic zone (18, 19). Also, similar to InhbaBK/- mice, the life span of Igf1 null mutant mice is markedly compromised (20). Because of these phenotypic similarities, we compared the serum IGF-I levels of adult wild-type and InhbaBK/BK mice. InhbaBK/BK mice had significantly lower serum IGF-I levels than wild-type mice (356.67 ± 36.02 ng/ml, vs. 810.83 ± 60.25 ng/ml, n = 68, P = 0.0001 for InhbaBK/BK and wild type, respectively). These results suggested a relationship between activin signaling and the regulation of IGF-I production, release, and/or bioavailability as possible mechanisms for the growth deficiencies of the InhbaBK knock-in mice. Because the liver is the primary source of circulating IGF-I (21), we next examined IGF-I mRNA levels in tissues from wild-type, InhbaBK/BK, and InhbaBK/- mice (Fig. 2
, A and B). Igf1 mRNA levels of wild-type, InhbaBK/BK and InhbaBK/- mice did not differ significantly in skeletal muscle, brain, spleen, and kidney; however, skeletal muscle showed a downward trend of Igf1 mRNA production and the liver showed a significant reduction of Igf1 mRNA from the wild-type levels in both InhbaBK/BK, and InhbaBK/- mice, corresponding to the dose of the InhbaBK allele (Fig. 2B
).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 2. RNAse Protection Assay (RPA) of IGF-I RNA from Tissues of Wild-type (WT), InhbaBK/BK (BK/BK), and InhbaBK/- (BK/-) Mice
Representative samples from mice of each genotype are shown for each tissue. A ß actin probe served as an internal control in all RPA experiments. The specific activities of both actin and IGF-I probes were different for liver RNA samples to adjust for the relative abundance of the Igf1 target RNA. H, Quantification of Igf1 mRNA. Igf1 mRNA levels were determined by RPA and PhosphorImager (Molecular Dynamics) analysis and are presented as a ratio to ß-actin mRNA. Note the decrease in Igf1 mRNA levels when mice of different genotypes are compared, restricted to liver and skeletal muscle. Asterisks denote significant differences from WT values.
|
|
Major Urinary Protein and GH Levels Are Normal in InhbaBK/BK Mice
Circulating GH exhibits gender-specific patterns of secretion in many species, including the mouse (22, 23). Although mean serum GH levels are comparable for both sexes, males release GH in a pulsatile fashion with high peaks of production at 34 h intervals followed by a rapid return to nearly undetectable levels (22, 24). In contrast, females have higher basal levels than males and exhibit much less variability. These differences in GH secretory patterning influence liver gene expression in a gender-specific fashion (22, 24).
Major urinary proteins (MUPs) are a highly heterogeneous group of proteins produced by the liver that bind to volatile pheremones. In rodents, they play important roles in individual recognition, territorial marking, and social behavior (25). They are the most abundant urinary proteins, and males excrete much higher levels than females. This gender-dependent difference is attributed to the secretory patterns of GH (24). Thus, MUPs are gender-specific indicators of GH secretion. To assess the relationship between GH secretion and the growth deficiency of InhbaBK/BK mice, we examined MUP production in 12-wk-old wild-type, InhbaBK/+, and InhbaBK/BK mice, coinciding with the age that MUP levels reach their peak. If the pattern of secretion for GH has been disrupted in InhbaBK/BK male mice, we reasoned that production of MUPs would be adversely affected in males, similar to male mice with pituitary GH deficiency (24). However, no decrease in MUP production was observed in InhbaBK/BK mice (Fig. 3A
), consistent with a normal male pattern of GH secretion. To determine directly if GH production or release was affected in InhbaBK/BK mice, we also measured serum and pituitary GH levels (Fig. 3
, B and C). No significant differences in plasma or pituitary GH levels were identified when comparing wild-type, InhbaBK/+, and InhbaBK/BK mice, consistent with normal GH production and release.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3. MUP and GH Levels in InhbaBK Knock-In Mice
A, Coomassie Blue staining of MUPs from wild-type (WT) InhbaBK/+ (BK/+) and InhbaBK/BK (BK/BK) mice. Note the high level of MUPs in the urine of male mice relative to females and normal levels of this protein in both male and female InhbaBK/BK mice relative to controls. Each of the panels shown is from the same gel. B, Serum GH levels of WT, InhbaBK/+, and InhbaBK/BK mice. No significant differences were observed in GH levels among the experimental groups (n =12), P > 0.1. C, Total GH content of pituitary glands of 6-wk-old wild-type and InhbaBK/BK mice. The values were not significantly different between the two groups (n = 67), P = 0.23.
|
|
Analysis of Liver and Kidney Function in InhbaBK Mice
Diminished liver mass, hepatocellular injury, and apoptosis occur when activins are administered exogenously in vivo and in vitro (26, 27) and in genetic models which lead to activin excess, such as inhibin-deficient mice (28). Furthermore, both type I and type II activin receptors are expressed in the liver (29, 30). These observations implicate activins as important regulators of hepatocellular function. However, relatively little is known regarding the effects of activin A signaling deficiency on the liver. Thus, we analyzed hepatic cytoarchitecture and function of InhbaBK/BK and InhbaBK/- mice. Although livers from 8-month old InhbaBK/BK and 3-wk-old InhbaBK/- mice were disproportionately small relative to wild type, they showed no histopathology when compared with age matched wild-type controls (Fig. 4
, AD). Importantly, none of the characteristic features of activin overexpression (centrilobular apoptosis and chronic lymphocytic inflammation) were observed. Measurement of the liver enzymes, aspartate aminotransferase (AST), and alanine aminotransferase (ALT), and albumin was carried out to assess hepatocellular integrity and synthetic hepatic function, respectively (Table 1
). Although no significant abnormalities in hepatic enzyme levels were observed among the experimental groups, plasma from InhbaBK/- mice showed a trend toward higher liver enzyme levels and both InhbaBK/BK and InhbaBK/- mice had significantly lower albumin levels than control mice (Table 1
).

View larger version (145K):
[in this window]
[in a new window]
|
Fig. 4. Hepatic and Renal Histology of InhbaBK Mice
A, Liver histology 8-month-old wild-type (WT) mouse. Two distinct central veins (C) are evident, as well as a clearly delineated portal canal (P) with a portal vein, adjacent bile duct (closed arrowhead), and hepatic artery. Basophilic hepatocyte nuclei contain prominent nucleoli. Hepatocytes are uniform in appearance with plentiful glycogen, confirmed by PAS staining (not shown). The differences in the prominence of the sinusoidal spaces among the sections are due to variability in the efficiency of perfusion of the vascular system from mouse to mouse. B, Liver from an 8-month-old InhbaBK/BK mouse (BK/BK). The hepatocellular architecture is comparable to the age-matched wild-type control mouse. One portal canal and two central veins are labeled. No inflammatory infiltrates were identified and abundant glycogen was present. C, Liver histology of a 21-d-old wild-type mouse demonstrating normal cytoarchitecture. The structural organization is similar to the adult, except hepatocyte volume and nuclear size is smaller in 21-d-old mice. D, Liver from a 21-d-old InhbaBK/- mouse. The structural organization of the liver is preserved relative to control mice. Grossly, the livers are smaller than those of wild-type mice, however, histologically the appearance is similar to the wild-type. No inflammatory infiltrates were present. A portal canal with portal vein (P), bile duct (closed arrowhead) and hepatic artery (open arrowhead) is shown. E, Low power view of kidney from an 8-month-old wild-type mouse demonstrating normal cytoarchitecture. Four glomeruli (G) are shown amid several proximal convoluted tubules (P). The proximal tubular epithelium has abundant microvilli on the lumenal surface, giving the lumen a foamy appearance. Two distal convoluted tubules (D) are shown that are comprised of cuboidal epithelium that lacks microvilli. The cells have dark basophilic nuclei. F, histology from kidney of an 8-month-old InhbaBK/BK mouse. No obvious differences are observed when compared with the wild-type mouse. Proximal and distal tubules and glomeruli are shown. G, Low power view of kidney section from a 21-d-old wild-type mouse. Note that the lumenal structure is not as well developed as in adult mice, making it more difficult to recognize specific tubule types. The glomeruli, though abundant, are generally smaller that in adult mice, giving the appearance of highly concentrated nuclei. H, Kidney section from 21-d-old InhbaBK/- mouse. Several glomeruli are shown. A large branch of the arterial circulation (A) is shown, giving rise to an afferent arteriole entering the glomerulus at the vascular pole (V). Opposite this is the urinary pole (U), which is continuous with the proximal tubule. No structural abnormalities were identified from the several sections that were compared from these mice and controls.
|
|
Expression of both activins ßA and ßB and the type II activin receptor have been demonstrated in the kidney (31, 32), and activins have been implicated as inhibitors of branching morphogenesis and glomerular development (for review, see Ref. 33). Furthermore, chronic renal insufficiency is associated with small stature through a complex mechanism by which uremia leads to decreased growth factor sensitivity and production (34). Therefore, we examined the kidneys of InhbaBK/BK and InhbaBK/- mice for histopathology (Fig. 4
, EH). We also analyzed blood and urine from these mice for evidence of renal dysfunction (Tables 1
and 2
). In contrast to our findings in the liver, the kidneys of InhbaBK/BK and InhbaBK/- mice were not disproportionately small when compared with the kidneys of wild-type mice. Histologically, no apparent differences were observed when comparing wild-type and InhbaBK/BK mice at 8 months of age or wild-type and InhbaBK/- mice at 21 d (Fig. 4
, EH). The glomeruli were well formed and of appropriate size, number, and distribution. Glomeruli were counted, and no significant differences were identified between 21-d-old wild-type and InhbaBK/- mice (22.71 ± 2.02 vs. 23.11 ± 0.86 glomeruli per low power field, respectively, n = 79). All segments of the tubule were present and exhibited normal architecture histologically. No inflammation or other obvious pathology was evident. Analysis of creatinine in plasma showed no differences among the experimental groups; however, a significant difference in blood urea nitrogen (BUN) was observed, with higher values for InhbaBK/BK mice relative to controls (Table 1
). InhbaBK/- mice also showed a trend toward higher BUN values, but the difference was not statistically significant. Both chemical and microscopic urinalyses were carried out; however, no significant differences were identified among the groups (Table 2
).
Endogenous Activin ßB Provides Functional Support in Maintaining Growth and Life Span
To further assess the roles of activin ßA and ßB spatiotemporal expression patterns, specific activin ligand/receptor interactions, and gene dosage in activin signaling, we produced double mutant mice with the InhbaBK allele and the Inhbb null allele (Fig. 5
). Because Inhba+/+, Inhbb-/- mice grow normally (15, 16), we predicted that InhbaBK/BK, Inhbb-/- mice would have phenotypes that were similar to those of InhbaBK/BK mice, possibly also with eyelid closure defects if the activin ßB protein produced from the InhbaBK allele was not sufficient to rescue the open eye phenotype of Inhbb-null mutant mice. Double heterozygous mice (InhbaBK/+, Inhbb+/-) were mated, and the genotypes of the offspring were determined at weaning (
3 wk of age). We expected 6.25% of the 238 mice analyzed from these matings to have the genotype, InhbaBK/BK, Inhbb-/-. However, no mice with this genotype were identified in this group, although other genotypes were represented at the predicted Mendelian ratios (Table 3
). Therefore, litters were subsequently examined during the first few days of life to look for pups with eyelid closure defects, the characteristic phenotype of Inhba+/+, Inhbb-/- mice (15, 16), and for delayed hair growth and somatic growth, phenotypic features of InhbaBK/BK, Inhbb+/+ mice (17). Several newborn pups had eyelid closure defects, and the sizes of the pups, with or without eyelid defects, differed considerably (Fig. 6
). Small mice with delayed hair growth and eyelid closure defects were identified in the postnatal period, corresponding to the genotype, InhbaBK/BK, Inhbb-/- (Fig. 6
). Thus, activin ßB produced from the InhbaBK allele is not sufficient to rescue the open eye phenotype of Inhba+/+, Inhbb-/- mice.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5. Genotype Analysis of Double Mutant Mice
A, The mature domain of activin ßA protein is encoded entirely within exon 2 (E2). The pertinent region of the wild-type Inhba sequence is shown for comparison. We have introduced a knock-in allele (InhbaBK) at the Inhba locus that is comprised of a hybrid exon 2 sequence, which encodes 2/3 of the activin ßA propeptide (gray box), and the mature domain of activin ßB (black box). The knock-in allele directs the production of activin ßB protein in sites where activin ßA is normally expressed. Like activin ßA, the wild-type activin ßB gene (Inhbb) also contains 2 exons. Functional inactivation of the gene was achieved by replacing exon 1 (E1) sequence encoding the translation initiation codon and the signal peptide with a Pgk-neo expression cassette (neo), producing the null allele. The mutation results in the inability to produce a functional activin ßB protein (15 ). B, Southern blot analysis of genomic tail DNA from mutant mice. The wild-type (Inhba) and mutant (InhbaBK) alleles are distinguishable by EcoRI (R) digestion followed by hybridization with a 3' probe (top panel). Wild-type (Inhbb) and null (Inhbb-) alleles at the Inhbb locus are distinguishable by digestion with BamHI (B) and hybridization with a 3' probe (bottom panel).
|
|

View larger version (108K):
[in this window]
[in a new window]
|
Fig. 6. Phenotypes of Inhba, Inhbb Double Mutant Mice
A, Four double mutant littermates at postnatal d 4 (P4), lateral view. All mice with at least one copy of the wild-type Inhba allele have normal growth and development. Mice that are homozygous for the InhbaBK knock-in allele have growth deficiencies that are influenced by gene dosage at the Inhbb locus. Left to right, InhbaBK/+, Inhbb+/-; InhbaBK/+,Inhbb-/-; InhbaBK/BK, Inhbb+/-; InhbaBK/BK, Inhbb-/-. B, Three double mutant littermates, P21. Although the InhbaBK/+, Inhbb+/- mouse (center) grows normally, both InhbaBK/BK, Inhbb+/- (left) and InhbaBK/BK, Inhbb-/- (right) mice have somatic growth deficiencies. C, Newborn InhbaBK/+, Inhbb-/- mouse (left) with failure of eyelid closure (open arrowhead). Contrast the appearance of the eye to that of a normal InhbaBK/+, Inhbb+/- littermate (right). D, InhbaBK/BK, Inhbb-/- mouse at P2. Notice the opaque appearance of the eye relative to the mouse in (C) as mechanical irritation of the cornea results in epithelial thickening (open arrowhead). Note also the thin translucent skin through which underlying vessels (filled arrowheads) and eye pigment are easily visible, in contrast to the newborn mice in (C).
|
|
In contrast to the 58% survival for InhbaBK/BK, Inhbb+/+mice at 26 wk (Fig. 7B
), InhbaBK/BK, Inhbb-/- mice demonstrated 50% survival at 3 d. An additional 25% expired by 2.5 wk. At P14, the surviving InhbaBK/BK, Inhbb-/- mice were 5060% the weight of their normal littermates (Fig. 7A
) and had nearly identical phenotypes as InhbaBK/- mice that we have described previously (17) (i.e. small, cachectic appearance, prominent external genitalia, paucity of hair in the genital area, and growth plate abnormalities (Fig. 6B
). Thus, the life span of InhbaBK/BK, Inhbb-/-mice was markedly less than that of InhbaBK/BK, Inhbb+/+mice.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 7. Growth and Survival of Wild-Type (WT) and Mutant Mice
A, Mean weights of male and female WT (n =1520), InhbaBK/BK, Inhbb+/+ (n = 2534), InhbaBK/BK, Inhbb+/- (n = 1223), and InhbaBK/BK, Inhbb-/- (n = 45), mice were determined for at least 21d. All mice that were homozygous for the InhbaBK allele demonstrated growth deficiencies relative to wild-type mice, and this deficiency was further exacerbated by progressively decreasing gene dose at the Inhbb locus. B, The survival of male and female mutant mice was examined weekly over a 26-wk period. InhbaBK/+, Inhbb+/- mice had normal survival, but the survival of all mice homozygous for the InhbaBK allele was dramatically influenced by gene dose at the Inhbb locus. InhbaBK/+, Inhbb+/- (n = 2325), InhbaBK/BK, Inhbb+/+ (n = 910), InhbaBK/BK, Inhbb+/- (n = 1223), and InhbaBK/Bk, Inhbb-/- (n = 45). Only mice that lived for at least 1 wk were included in the survival study.
|
|
Next, we examined InhbaBK/BK, Inhbb+/-mice to determine if their physical characteristics were distinguishable from InhbaBK/BK, Inhbb+/+ and InhbaBK/BK, Inhbb-/- mice. Although both double mutant InhbaBK/+, Inhbb+/- and InhbaBK/+, Inhbb-/-mice grew normally due to the presence of one wild-type Inhba allele, InhbaBK/BK, Inhbb+/- mice had an intermediate growth deficiency to InhbaBK/BK, Inhbb+/+ and InhbaBK/BK, Inhbb-/- mice (Figs. 6
, A and B, and 7A
) and also had an intermediate 50% survival rate of 6 wk (Fig. 7B
).
 |
DISCUSSION
|
---|
Because the growth of multiple tissues and organs not known to produce activins directly in substantial quantities are affected in our knock-in mice (17) (and unpublished observations), it was conceivable that the downstream effects of the InhbaBK allele were due to secondary effects on GH production or release. However, we argue that it is unlikely that the poor growth of the knock-in mice is a direct consequence of GH deficiency. Several lines of evidence support this notion. First, the growth deficiencies of both InhbaBK/BK and InhbaBK/- mice are evident shortly after birth, reflecting a very early effect of the mutation (Figs. 6
and 7
). In contrast, mice with either a homozygous null mutation of the GH receptor (35) or with functional ablation of GH-producing cells (36) do not manifest growth deficiencies until P17-P21. Secondly, activin ßA has been reported to inhibit, rather than augment, GH release from cultured pituitary somatotrophs (37, 38) and so a hypomorphic activin protein produced from the InhbaBK allele would likely be a less effective inhibitor than the wild-type activin ßA protein. Next, activin ßB is the predominant activin produced in the pituitary glands of wild-type mice (31, 39), yet activin ßB null mutant mice grow normally (15), suggesting that activin ßB has little if any net effect on GH release from the pituitary gland in vivo. Finally, our measurements of serum and pituitary GH levels, and assessment of major urinary proteins in InhbaBK/BK growth-deficient mice showed no significant differences from wild-type mice. Taken together, these observations indicate that GH deficiency is not the mechanism by which growth is adversely affected in InhbaBK/BK mice. Thus, it is likely that the growth deficiencies of InhbaBK/BK and InhbaBK/- mice are either due to direct consequences of compromised activin signaling, such as diminished IGF-I production, indirectly due to the compromised function of major organ systems, or both.
IGF-I and IGF-II are important contributors to prenatal and postnatal growth (18, 40, 41). Igf1 knockout mice have birth weights that are approximately 60% of normal (41). In addition, IGF-I is essential for normal life span, because 3290% of Igf1-/- mice die within the first few weeks of life, depending on the genetic background (41). Although the specific cause of death of Igf1 knockout mice is also not clear, a number of morphologic abnormalities have been described in IGF-I receptor knockout mice that all die in the newborn period. These abnormalities include respiratory failure at birth, hypoplasia of muscle and other tissues, thin translucent skin, and delayed bone development (41). Interestingly, all of these abnormalities have been observed in mice with mutations at either the activin ßA locus (12, 17), or in the gene encoding follistatin, an activin regulatory protein (42). Therefore, the phenotypic similarities between activin and IGF-I mutants may provide important clues as to the cause(s) of the decreased life span of InhbaBK knock-in mice. Although the longevity of InhbaBK mice is reduced, the specific cause of death, presumably linked to activin ßA deficiency, remains uncertain. We have previously demonstrated hypoglycemia and anemia in InhbaBK/-mice (17). In this paper, our evaluation of hepatic and renal function showed a modest elevation of liver enzymes in InhbaBK/-mice, and both InhbaBK/BK and InhbaBK/-mice had lower albumin levels than control mice despite normal histology (Table 1
). The reason for the low albumin levels is unclear, possibly reflecting the relatively poor nutritional status of these mice, abnormal loss of protein in the urine or compromised synthetic function of the liver. The normal levels of major urinary proteins by SDS-PAGE and absence of proteinuria from urinalyses argue against excessive losses from the kidneys. In light of modestly elevated liver enzymes, however, it is possible that both compromised hepatic function and nutritional status play a role. InhbaBK/BK mice also had significant elevations of blood urea nitrogen, suggesting possible renal dysfunction. Alternatively, this finding is consistent with dehydration. Normal renal cytoarchitecture, urinalyses, and creatinine values favor the second possibility, although more comprehensive studies of renal function will be required to address the question definitively. Finally, the quantity of adipose tissue in InhbaBK/- mice is markedly less than in controls (Brown, C. W., unpublished observations). This finding suggests a possible disruption of metabolism in favor of a catabolic state, due to either impaired energy storage mechanisms or increased breakdown of energy stores. The hypoglycemia that has previously been observed in InhbaBK/- mice would be consistent with this assertion. Thus, it is possible that activin deficiency also influences the metabolic rate or biochemical factors that play important roles in energy metabolism. Experiments to address these possibilities are in progress.
In summary, the InhbaBK knock-in mice have exhibited modest abnormalities of many different analytes, suggesting that the shortened life span of InhbaBK/BK and InhbaBK/- mice is likely due to the collective effects of reduced activin signaling on many different organ systems.
Activin signaling is important in a variety of biological processes that involve concomitant synergistic or inhibitory signaling pathways, including red blood cell production (6, 43), gonadotropin production (3, 44), and inflammation (45). Thus, it is plausible that activins modulate IGF-I receptor signaling and that IGF-I and other pathways have reciprocal effects on activin signaling. Consistent with this notion, cultured rat ovarian granulosa cells require IGF-I to maintain activin-dependent inhibin-
expression (46). Additional experiments will be required, however, to clarify the relationships between activins and these other pathways, and genetic approaches can be used to test the hypothesis that these pathways interact by comparing phenotypic characteristics of single and double mutant mice.
Serum IGF-I levels were 56% lower in InhbaBK/BK mice than controls, and we observed tissue selective differences in IGF-I mRNA levels in the liver, the major source for IGF-I in the circulation. This suggests either that activin signaling provides a supportive function to IGF-I production or release from the liver to maintain normal somatic growth, in keeping with the classical somatomedin hypothesis, or that it plays a direct role in activating genes that are important contributors to somatic growth, independent of the GH/IGF-I signaling pathways. The observation that a significant decrease of mRNA encoding IGF-I was observed only in the livers of InhbaBK growth-deficient mice may reflect a requirement for a minimum threshold of activin signaling in the liver to maintain high level IGF-I production at the transcriptional and/or posttranscriptional levels.
We have shown previously that production of activin ßB subunits from the Inhba locus influences growth and survival, and the degree of influence is determined by both the dosage and bioactivity of the peptides encoded by these alleles (17). Here, we examined whether the Inhbb locus could further influence growth and survival. The phenotypic similarity of the InhbaBK/BK, Inhbb-/- and InhbaBK/-, Inhbb+/+ mice suggests that not only must a critical threshold of activin signaling be maintained for normal growth and life span, but also that both activin ßA and activin ßB have the potential to participate in these complicated biological processes (Fig. 8
). However, these phenotypic similarities do not necessarily imply a common mechanism for growth deficiency or decreased life span for single and double mutant mice because double mutant mice have not been evaluated systematically in our studies. Nevertheless, the biopotency of activin ßA in vivo must exceed that of activin ßB, because all mice with at least one copy of the wild-type Inhba allele grow normally and have a normal life span, irrespective of the genotype at the Inhbb locus. In contrast, mice with four alleles that produce activin ßB (i.e. InhbaBK/BK, Inhbb+/+ mice) are not phenotypically normal despite the fact that normal levels of activin ßB peptides are produced from the InhbaBK allele in the correct spatiotemporal fashion as we have shown previously (17). Thus, in both InhbaBK/BK and InhbaBK/- mice, the activin ßB protein produced from the endogenous Inhbb locus is an important contributor to activin signaling. However, its contribution to the maintenance of somatic growth and life span is not required in mice with at least one normal copy of the Inhba allele.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 8. Hypothetical Model for Threshold Effects in Global Activin Signaling
The vertical dashed lines represent the minimum threshold of activin signaling that must be maintained for each process to occur normally. The superimposed gradient represents the influence of factors that antagonize activin signaling, such as follistatin or inhibins. In this model, as the intrinsic bioactivity of the total activin milieu is compromised, such as in all of the InhbaBK mice shown here, the delicate balance between activin signaling and antagonistic processes is disturbed, with antagonistic processes having the greatest influence in mice with the most compromised activin intrinsic activity (i.e. InhbaBK/BK, Inhbb-/- and InhbaBK/- mice). Note that the intrinsic biopotency of activin ßA as shown greatly exceeds that of activin ßB and that the bioactivity of activin ßB produced from the InhbaBK allele exceeds that produced from the Inhbb allele due to its broader pattern of distribution and/or its higher level of production from the Inhba locus relative to the Inhbb locus. Note also that this process is dynamic, because both levels and patterns of expression of the activins and their modulators change with time.
|
|
Simple differences in the total number of alleles that produce activin ßB protein cannot account entirely for the phenotypic differences observed among the double mutant mice, because distributing the same number of activin ßB-producing alleles among the Inhba and Inhbb loci in different combinations does not result in identical phenotypes. Two major explanations for this observation are that the Inhba locus is transcriptionally active in a greater number of postnatal cell types than the Inhbb locus (31, 47), as it is during embryonic development (48, 49, 50) and that in the normal condition, the activin ßA protein is transcribed, translated, or secreted at higher levels than activin ßB in most tissues (31, 47). In a global sense, these differences in pattern and/or quantity of expression should result in a greater sensitivity to Inhba mutations, because such changes would affect a proportionately larger number of cells and tissues and should have greater consequences with respect to total activin production. Another possibility is that misexpression of activin ßB from the InhbaBK allele affects the cellular milieu of factors, i.e. inhibins, follistatin, and type I and II receptor combinations, that regulate activin signaling (Fig. 8
). Finally, it is important to consider that the absence of activin ßA in InhbaBK/BK and InhbaBK/- mice results in a loss of activin ßA:ßB heterodimers that may play specific roles in activin signaling. However, it seems less likely that this deficiency would be a specific cause for the abnormalities of the InhbaBK mice because singly mutant Inhba+/+, Inhbb-/- mice also lack activin ßA:ßB heterodimers, yet have none of the phenotypic characteristics of InhbaBK knock-in mice. Also, no differences would be predicted between InhbaBK/BK and InhbaBK/- mice if the abnormal phenotypes were due to the specific effects of activin AB heterodimer deficiency.
In conclusion, we have demonstrated that both activins ßA and ßB can influence growth and survival, but the contribution of activin ßB to these processes is probably minimal in wild-type mice. However, if the biological activity of the activin ßA protein is compromised, as in our InhbaBK mutants, major organ systems are likely affected and activin ßB is enlisted to support activin ßAs roles in maintaining growth and survival, possibly, through its effects on IGF-I, but without significant effects on the synthesis, or release of GH.
 |
MATERIALS AND METHODS
|
---|
Histology and Growth Plate Analysis
Three-week-old mice were anesthetized in accordance with standard institutional protocols, observing the highest standards of humane animal care. The vascular system was then perfused with 0.9% NaCl followed by 10% neutral buffered formalin. Liver and kidneys were removed and placed in 10% neutral buffered formalin. The femoral/tibial joints were dissected, fixed in formalin overnight at room temperature, partially demineralized for 25 min in TBD-1 rapid decalcifier (Shandon, Inc., Pittsburgh, PA) then rinsed in PBS and formalin. All tissues were embedded in paraffin and sectioned (58 µm). Growth plates sectioned at the mid-coronal or mid-sagittal plane were stained with Massons trichrome as described (51). Sections from liver and kidney were stained with hematoxylin and eosin. For growth plate quantitative analysis, sections from the mid-sagittal plane were examined. Digital images were captured directly from the microscope using a x20 objective. After calibration to a micrometer scale, we measured growth plate thickness using Spot Image analysis software (version 3.3.2, Diagnostic Instruments, Inc., Sterling Heights, MI). Four different sections from at least two mice of each genotype were examined. Measurements of total growth plate, proliferative zone and hypertrophic zone thicknesses were taken at three to four positions across the growth plate and averaged. Measurements of cell area were obtained directly, also with Spot image analysis software, by tracing the cell perimeter. At least 12 cells of the hypertrophic zone were measured per section, and mean values for each genotype were obtained.
Laboratory Analysis of Liver and Kidney Function
Blood samples were obtained from 129/SvEv-C57BL/6 hybrid mice under isoflurane anesthesia by retro-orbital bleed for adult mice and by cardiac puncture in 21-d-old mice. Serum was stored at -20 C until analysis. ALT, AST, albumin, BUN, creatinine measurements and urinalyses were done by the Comparative Pathology Laboratory (Baylor College of Medicine). Serum analyses were done using a COBAS Integra 400 plus System (Roche Molecular Biochemicals, Indianapolis, IN). Urinalysis was carried out using Multistix 10SG (Fisher Scientific, Pittsburgh, PA).
Assessment of Major Urinary Proteins
Urine was collected from 12-wk-old male and female mice by startle response over a weigh boat. All samples were collected in the early evening (
1800 h) and were frozen at -80 C until use. The samples were thawed, centrifuged at 14,000 rpm for 1 min, and 2 µl of urine supernatant was then added to 18 µl of running buffer [70 mM Tris (pH 6.8), 2.2% sodium dodecyl sulfate (SDS), 5.55% glycerol, 0.055% bromophenol blue]. Samples were then heated to 95 C for 3 min, spun briefly, and 10 µl were loaded and electrophoresed through precast 12% Tris-HCl, polyacrylamide gels (Bio-Rad, Hercules, CA), using Tris/glycine/SDS buffer (Bio-Rad). After electrophoresis, duplicate gels were stained with Coomassie blue. Major urinary proteins were the most abundant proteins on the gels, migrating to a position of approximately 20 kDa.
GH Measurements
Plasma was obtained from 42-d-old mice. Samples were collected in the early evening (
1800 h) to minimize the effects of diurnal variation, and were stored at -20 C until use. For pituitary extracts, 6-wk-old mice were euthanized and whole pituitary glands were homogenized in 100 µl of ice-cold Nonidet P-40 extraction buffer (150 mM NaCl, 1% Nonidet P-40, 50 mM Tris 8.0, 1.5 µg/ml leupeptin, 1.5 mM phenylmethylsulfonyl fluoride). The suspensions were frozen at -20 C until use. Serum GH was measured in undiluted samples using SPI-BIO rat GH enzyme immunoassay kits (Cayman Chemical Co., Ann Arbor, MI; distributor), according to the manufacturers instructions. Pituitary lysates were diluted 100-fold in assay buffer, and the nanograms of GH present in the equivalent of 1 µl of lysate were determined by RIA according to the manufacturers instructions (rat GH [125I] assay system with magnetic separation (RPA551), Amersham Pharmacia Biotech, Piscataway, NJ). The number of measured nanograms per sample was then multiplied by the total volume of the lysate (100 µl) to determine the total pituitary GH content.
Serum IGF-I Measurement
Sera were collected from 69 wk of age, anesthetized mice by closed cardiac puncture. IGF-I levels were measured by RIA using a solid phase RIA kit according to the manufacturers instructions (Diagnostic Systems Laboratories, Inc., Webster, TX).
Ribonuclease (RNAse) Protection Assays
RNAse protection assays, using the RPA III Ribonuclease Protection Assay Kits (Ambion, Austin, TX), were carried out on total cellular RNA extracted from 3-wk mouse skeletal muscle, kidney, spleen, brain, and liver. Antisense radiolabeled RNA probes were produced from linearized plasmid DNA using T7 RNA polymerase (Promega, Madison, WI). The specific activities of the probes were calculated after trichloroacetic acid precipitation, and care was taken to ensure that the probes were present in molar excess to target RNA. RNA Stat-60 was used to prepare the total cellular RNA, as described by the manufacturer (Leedo Medical Laboratories, Houston, TX). Twenty micrograms of RNA from skeletal muscle, kidney, brain, and spleen or 10 µg from liver were used per hybridization. Hybridizations were performed for 14 h at 42 C and RNAse digestion was carried out using 0.5 U RNAse A and 20 U RNAse T1 per sample for 30 min at 37 C. The protected products were electrophoresed through 5% nondenaturing polyacrylamide gels. Dried gels were exposed to X-OMAT film (Kodak), and then to Phosphorimaging plates for quantification of bands. Counts were determined using a Storm 860 analyzer (Molecular Dynamics, Sunnyvale, CA). Image Quant software (Molecular Dynamics) was used to quantify individual bands relative to an internal ß actin standard.
Production of Double Mutant Mice
Female mice heterozygous for the activin ßB knock-in allele (InhbaBK/+) were mated with male activin ßB homozygous null mice (Inhbb-/-). The double heterozygous offspring (InhbaBK/+, Inhbb+/-) were mated to produce mice with different combinations of alleles at the Inhba and Inhbb loci. To increase the likelihood that double homozygous mutants would be produced, we also established matings between InhbaBK/+, Inhbb-/-males and InhbaBK/+, Inhbb+/- females. The construct assembly and targeting strategy for the Inhbb null allele (Inhbbtm1Jae, herein, Inhbb-) (15), Inhba null allele (Inhbatm1Zuk, herein, Inhba-) (12) and activin ßB knock-in allele (InhbaBK) (17) have been described previously. The InhbaBK allele contains a hybrid activin ßA/ßB exon 2 that was constructed by PCR. The generation of the construct and the production of InhbaBK mutant mice have been described (17).
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Dr. T. Rajendra Kumar for critical review of the manuscript, and Drs. Anne Vassalli and Rudolph Jaenisch for the gift of the inhibin/activin ßB mutant mice.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants HD32067 (to M.M.M.), HD01156, and HD27823 (to C.W.B.), and the Robert Wood Johnson Foundation. This work was also supported in part by Research Grant 5-FY01-482 from the March of Dimes Birth Defects Foundation. C.W.B. is a recipient of a Burroughs Wellcome Fund Career Award in the Biomedical Sciences.
Abbreviations: ALT, Alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; MUP, major urinary proteins; RNAse, ribonuclease; SDS, sodium dodecyl sulfate.
Received for publication February 11, 2003.
Accepted for publication September 22, 2003.
 |
REFERENCES
|
---|
- Shi Y, Massague J 2003 Mechanisms of TGF-ß signaling from cell membrane to the nucleus. Cell 113:685700[Medline]
- Chang H, Brown CW, Matzuk MM 2002 Genetic analysis of the mammalian transforming growth factor-ß superfamily. Endocr Rev 23:787823[Abstract/Free Full Text]
- de Kretser DM, Robertson DM 1989 The isolation and physiology of inhibin and related proteins. Biol Reprod 40:3347[Abstract]
- Yamaoka T, Idehara C, Yano M, Matsushita T, Yamada T, Ii S, Moritani M, Hata J, Sugino H, Noji S, Itakura M 1998 Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants. J Clin Invest 102:294301[Abstract/Free Full Text]
- Molloy CJ, Taylor DS, Pawlowski JE 1999 Novel cardiovascular actions of the activins. J Endocrinol 161:179185[Abstract/Free Full Text]
- Yu J, Shao L, Lemas V, Yu AL, Vaughan J, Rivier J, Vale W 1987 Importance of FSH-releasing protein and inhibin in erythrodifferentiation. Nature 330:765[CrossRef][Medline]
- de Kretser DM, Hedger MP, Phillips DJ 1999 Activin A and follistatin: their role in the acute phase reaction and inflammation. J Endocrinol 161:195198[Free Full Text]
- Munz B, Hubner G, Tretter Y, Alzheimer C, Werner S 1999 A novel role of activin in inflammation and repair. J Endocrinol 161:187193[Free Full Text]
- Aono T, Sugino H, Vale WW, eds. 1997 Inhibin, activin and follistatin regulatory functions in system and cell biology. New York: Springer-Verlag
- de Kretser DM, Meinhardt A, Meehan T, Phillips DJ, OBryan MK, Loveland KA 2000 The roles of inhibin and related peptides in gonadal function. Mol Cell Endocrinol 161:4346[CrossRef][Medline]
- McPherron AC, Lee S-J 1993 GDF-3 and GDF-9: two new members of the transforming growth factor-ß superfamily containing a novel pattern of cysteines. J Biol Chem 268:34443449[Abstract/Free Full Text]
- Matzuk MM, Kumar TR, Vassalli A, Bickenbach JR, Roop DR, Jaenisch R, Bradley A 1995 Functional analysis of activins during mammalian development. Nature 374:354356[CrossRef][Medline]
- Ferguson CA, Tucker AS, Christensen L, Lau AL, Matzuk MM, Sharpe PT 1998 Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition. Genes Dev 12:26362649[Abstract/Free Full Text]
- Jhaveri S, Erzurumlu RS, Chiaia N, Kumar TR, Matzuk MM 1998 Defective whisker follicles and altered brainstem patterns in activin and follistatin knockout mice. Mol Cell Neurosci 12:206219[CrossRef][Medline]
- Vassalli A, Matzuk MM, Gardner HA, Lee KF, Jaenisch R 1994 Activin/inhibin ß B subunit gene disruption leads to defects in eyelid development and female reproduction. Genes Dev 8:414427[Abstract]
- Schrewe H, Gendron-Maguire M, Harbison ML, Gridley T 1994 Mice homozygous for a null mutation of activin ßB are viable and fertile. Mech Dev 47:4351[CrossRef][Medline]
- Brown CW, Houston-Hawkins DE, Woodruff TK, Matzuk MM 2000 Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions. Nat Genet 25:453457[CrossRef][Medline]
- Baker J, Liu JP, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:7382[Medline]
- Wang J, Zhou J, Bondy CA 1999 Igf1 promotes longitudinal bone growth by insulin-like actions augmenting chondrocyte hypertrophy. FASEB J 13:19851990[Abstract/Free Full Text]
- Powell-Braxton L, Hollingshead P, Warburton C, Dowd M, Pitts-Meek S, Dalton D, Gillett N, Stewart TA 1993 IGF-I is required for normal embryonic growth in mice. Genes Dev 7:26092617[Abstract]
- Sjogren K, Liu JL, Blad K, Skrtic S, Vidal O, Wallenius V, LeRoith D, Tornell J, Isaksson OG, Jansson JO, Ohlsson C 1999 Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proc Natl Acad Sci USA 96:70887092[Abstract/Free Full Text]
- Waxman DJ, Pampori NA, Ram PA, Agrawal AK, Shapiro BH 1991 Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450. Proc Natl Acad Sci USA 88:68686872[Abstract]
- Waxman DJ, Ram PA, Park S-H, Choi HK 1995 Intermittent plasma growth hormone triggers tyrosine phosphorylation and nuclear translocation of a liver-expressed, stat 5-related DNA binding protein. J Biol Chem 270:1326213270[Abstract/Free Full Text]
- Norstedt G, Palmiter R 1984 Secretory rhythm of growth hormone regulates sexual differentiation of mouse liver. Cell 36:805812[Medline]
- Hurst JL, Payne CE, Nevison CM, Marie AD, Humphries RE, Robertson DHL, Cavaggioni A, Beynon RJ 2001 Individual recognition in mice mediated by major urinary proteins. Nature 414:631634[CrossRef][Medline]
- Schwall RH, Robbins K, Jardieu P, Chang L, Lai C, Terrell TG 1993 Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology 18:347356[Medline]
- Hully JR, Chang L, Schwall RH, Widmer HR, Terrell TG, Gillett NA 1994 Induction of apoptosis in the murine liver with recombinant human activin A. Hepatology 20:854862[Medline]
- Matzuk MM, Finegold MJ, Mather JP, Krummen L, Lu H, Bradley A 1994 Development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice. Proc Natl Acad Sci USA 91:88178821[Abstract]
- Coerver KA, Woodruff TK, Finegold MJ, Mather J, Bradley A, Matzuk MM 1996 Activin signaling through activin receptor type II causes the cachexia-like symptoms in inhibin-deficient mice. Mol Endocrinol 10:534543[Abstract]
- Chen W, Woodruff TK, Mayo KE 2000 Activin A-induced HepG2 liver cell apoptosis: involvement of activin receptors and smad proteins. Endocrinology 141:12631272[Abstract/Free Full Text]
- Meunier H, Rivier C, Evans RM, Vale W 1988 Gonadal and extragonadal expression of inhibin
, ßA, and ßB subunits in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247251[Abstract]
- Tuuri T, Eramaa M, Hilden K, Ritvos O 1994 The tissue distribution of activin ßA- and ßB-subunit and follistatin messenger ribonucleic acids suggests multiple sites of action for the activin-follistatin system during human development. J Clin Endocrinol Metab 78:15211524[Abstract]
- Ball E, Risbridger GP 2001 Activins as regulators of branching morphogenesis. Dev Biol 238:112[CrossRef][Medline]
- Rabkin R 2001 Growth factor insensitivity in renal failure. Ren Fail 23:291300[CrossRef]
- Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Cataldo L, Coschigamo K, Wagner TE, Baumann G, Kopchick JJ 1997 A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse). Proc Natl Acad Sci USA 94:1321513220[Abstract/Free Full Text]
- Behringer RR, Mathews LS, Palmiter RD, Brinster RL 1988 Dwarf mice produced by genetic ablation of growth hormone-expressing cells. Genes Dev 2:453461[Abstract]
- Bilezikjian LM, Corrigan AZ, Vale W 1990 Activin-A modulates growth hormone secretion from cultures of rat anterior pituitary cells. Endocrinology 126:23692376[Abstract]
- Billestrup N, Gonzalez-Manchon C, Potter E, Vale W 1990 Inhibition of somatotroph growth and growth hormone biosynthesis by activin in vitro. Mol Endocrinol 4:356362[Abstract]
- Roberts VJ, Peto CA, Vale W, Sawchenko PE 1992 Inhibin/activin subunits are costored with FSH and LH in secretory granules of the rat anterior pituitary gland. Neuroendocrinology 56:214224[Medline]
- Jones JI, Clemmons DR 1995 Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16:334[Medline]
- Liu JP, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75:5972[Medline]
- Matzuk MM, Lu N, Vogel H, Sellheyer K, Roop DR, Bradley A 1995 Multiple defects and perinatal death in mice deficient in follistatin. Nature 374:360363[CrossRef][Medline]
- Schwall R, Schmelzer CH, Matsuyama E, Mason AJ 1989 Multiple action of recombinant activin-A in vivo. Endocrinology 125:14201423[Abstract]
- Childs GV, Unabia G 1997 Cytochemical studies of the effects of activin on gonadotropin-releasing hormone (GnRH) binding by pituitary gonadotropes and growth hormone cells. J Histochem Cytochem 45:16031610[Abstract/Free Full Text]
- Brosh N, Sternberg D, Honigwachs-Shaanani J, Lee BC, Shav-Tal Y, Tzehoval E, Shulman LM, Toledo J, Hachman Y, Carmi P, Wen J, Sasse J, Horn F, Burstein Y, Zipoi D 1995 The plasmacytoma growth inhibitor restrictin-P is an antagonist of interleukin 6 and interleukin 11. Identification as a stroma-derived activin A. J Biol Chem 270:2959429600[Abstract/Free Full Text]
- Kubo T, Shimasaki S, Kim H, Li D, Erickson GF 1998 Activin-induced inhibin
-subunit production by rat granulosa cells requires endogenous insulin-like growth factor-I. Biol Reprod 58:712718[Abstract]
- Schneider O, Nau R, Michel U 2000 Comparative analysis of follistatin-, activin ßA- and activin ßB-mRNA steady-state levels in diverse porcine tissues by multiplex S1 nuclease analysis. Eur J Endocrinol 142:537544[Medline]
- Roberts VJ, Sawchenko PE, Vale W 1991 Expression of inhibin/activin subunit messenger ribonucleic acids during rat embryogenesis. Endocrinology 128:31223129[Abstract]
- Roberts VJ, Barth SL 1994 Expression of messenger ribonucleic acids encoding the inhibin/activin system during mid- and late-gestation rat embryogenesis. Endocrinology 134:914923[Abstract]
- Feijen A, Goumans MJ, van den Eijnden-van Raaij AJ 1994 Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development 120:36213637[Abstract/Free Full Text]
- Carson FL 1997 In: Histotechnology: a self-instructional text. Vol 1. 2nd ed. Chicago: ASCP Press