A novel GFP reporter mouse reveals Mustn1 expression in adult regenerating skeletal muscle, activated satellite cells and differentiating myoblasts.

AIM: Mustn1 has been implicated in myofusion as well as skeletal muscle growth and repair; however, the exact role and spatio-temporal expression of Mustn1 have yet to be fully defined. METHODS: Transgenic mice were generated with a 1512-bp sequence of the Mustn1 promoter directing the expression of GFP (Mustn1(PRO) -GFP). These mice were used to investigate the spatio-temporal expression of Mustn1(PRO) -GFP during skeletal muscle development and adult skeletal muscle repair, as well as various phases of the satellite cell lifespan (i.e. quiescence, activation, proliferation, differentiation). RESULTS: Mustn1(PRO) -GFP expression was observed within somites at embryonic day 12 and developing skeletal muscles at embryonic day 15 and 18. While uninjured adult tibialis anterior muscle displayed no detectable Mustn1(PRO) -GFP expression, cardiotoxin injury robustly elevated Mustn1(PRO) -GFP expression at 3 days post-injury with decreasing levels observed at 5 days and minimal, focal expression seen at 10 days. The expression of Mustn1(PRO) -GFP at 3 days post-injury consistently overlaid with MyoD although the strongest expression of Mustn1(PRO) -GFP was noted in newly formed myotubes that were expressing minimal levels of MyoD. By 5 days post-injury, Mustn1(PRO) -GFP overlaid in all myotubes expressing myogenin although cells were present expressing Mustn1(PRO) -GFP alone. The expression patterns of Mustn1(PRO) -GFP in regenerating muscle preceded the expression of desmin throughout the regenerative time course consistent with Mustn1 being upstream of this myogenic protein. Further, quiescent satellite cells located on freshly isolated, single myofibers rarely expressed Mustn1(PRO) -GFP, but within 24 h of isolation, all activated satellite cells expressed Mustn1(PRO) -GFP. Expression of Mustn1(PRO) -GFP in primary myoblasts diminished with prolonged time in proliferation media. However, in response to serum withdrawal, the expression of Mustn1(PRO) -GFP increased during myofusion (day 2) followed by declining expression thereafter. CONCLUSION: Mustn1(PRO) -GFP is expressed in activated satellite cells and myoblasts but continued time in proliferation media diminished Mustn1(PRO) -GFP expression. However, myoblasts exposed to serum withdrawal increased Mustn1(PRO) -GFP expression consistent with its demonstrated role in myofusion. The in vivo expression pattern of Mustn1 observed in regenerating and developing skeletal muscle is consistent with its presence in satellite cells and its critical role in myofusion.

Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene.

Our previous studies have demonstrated that promoter-green fluorescent protein (GFP) transgenes can be used to identify and isolate populations of cells at the preosteoblastic stage (pOBCol3.6GFP) and at the mature osteoblastic stage (pOBCol2.3GFP) in living primary bone cell cultures. This strategy forms the basis for appreciating the cellular heterogeneity of lineage and relating gene function to cell differentiation. A weakness of this approach was the lack of a selective marker for late osteoblasts and mature osteocytes in the mineralized matrix. In this study, we have examined the expression of DMP-1 mRNA in murine marrow stromal and calvarial osteoblast cultures, and in bone, and calvaria in vivo. Furthermore, we have generated transgenic mice utilizing a mouse DMP1 cis-regulatory system to drive GFP as a marker for living osteocytes. Transgene expression was directed to mineralized tissues and showed a high correlation with the expression of the endogenous gene. Osteocyte-restricted expression of GFP was observed in histological sections of femur and calvaria and in primary cell cultures. Generation of this transgenic model will facilitate studies of gene expression and biological functions in these terminally differentiated bone cells.

Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation.

Msx2 is believed to play a role in regulating bone development, particularly in sutures of cranial bone. In this study we investigated the effects of retroviral-mediated overexpression of Msx2 mRNA, in both sense and antisense orientations, on primary cultured chick calvarial osteoblasts. Unregulated overexpression of sense mRNA produced high levels of Msx2 protein throughout the culture period, preventing the expected fall as the cells differentiate. The continued high expression of Msx2 prevented osteoblastic differentiation and mineralization of the extracellular matrix. In contrast, expression of antisense Msx2 RNA decreased proliferation and accelerated differentiation. In other studies, we showed that the Msx2 promoter was widely expressed during the proliferative phase of mouse calvarial osteoblast cultures but was preferentially downregulated in osteoblastic nodules. These results support a model in which Msx2 prevents differentiation and stimulates proliferation of cells at the extreme ends of the osteogenic fronts of the calvariae, facilitating expansion of the skull and closure of the suture.

Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice.

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice.

Previous deletion studies using a series of COL1A1-CAT fusion genes have indicated that the 625 bp region of the COL1A1 upstream promoter between -2295 and -1670 bp is required for high levels of expression in bone, tendon, and skin of transgenic mice. To further define the important sequences within this region, a new series of deletion constructs extending to -1997, -1794, -1763, and -1719 bp has been analyzed in transgenic mice. Transgene activity, determined by measuring CAT activity in tissue extracts of 6- to 8-day-old transgenic mouse calvariae, remains high for all the new deletion constructs and drops to undetectable levels in calvariae containing the -1670 bp construct. These results indicate that the 49 bp region of the COL1A1 promoter between -1719 and -1670 bp is required for high COL1A1 expression in bone. Although deletion of the same region caused a substantial reduction of promoter activity in tail tendon, the construct extending to -1670 bp is still expressed in this tissue. However, further deletion of the promoter to -944 bp abolished activity in tendon. Gel mobility shift studies identified a protein in calvarial nuclear extracts that is not found in tendon nuclear extracts, which binds within this 49 bp region. Our study has delineated sequences in the COL1A1 promoter required for expression of the COL1A1 gene in high type I collagen-producing tissues, and suggests that different cis elements control expression of the COL1A1 gene in bone and tendon.

Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells.

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.

Multiple regulatory elements determine adrenocortical expression of steroid 21-hydroxylase.

Steroid 21-hydroxylase (21-OHase) is specifically expressed at high levels in the adrenal cortex, where it is required for the synthesis of mineralocorticoids and glucocorticoids. In this study, we have investigated the regulatory elements in the 21-OHase promoter region which contribute to the expression of this gene in Y1 adrenocortical cells. Eight potential regulatory elements in the 5'-flanking region of the 21-OHase gene were identified by DNase I footprinting and gel mobility shift experiments. Some of these footprints were produced by nuclear extracts from many cell lines, whereas other interactions were seen only when using nuclear extracts from Y1 adrenocortical and MA-10 Leydig tumor cells. Mutation of most of the elements markedly decreased the expression of a 21-OHase gene transfected into Y1 cells, thus documenting their functional importance for expression. Moreover, oligonucleotides containing the sequences of two related elements at -65 and -210, which share the heptamer AGGTCAG, increased the activity of a heterologous promoter in a Y1 cell-specific manner. Collectively, these results demonstrate that expression of 21-OHase in Y1 adrenocortical cells requires interactions among multiple cis-acting elements and regulatory proteins.

Identification and analysis of keratin polypeptides from isolated rat vaginal epithelium.

Under the influence of estradiol, the vaginal epithelium undergoes a process of growth and differentiation known as cornification. To analyze the biosynthetic changes that underlie this phenomenon in rats, it was necessary to first isolate the vaginal epithelium from hormonally stimulated animals. This was accomplished by two different methods: enzymatic digestion with a trypsin/pancreatin solution, which produced a disaggregated population of cells from the living epithelial layers; or disruption of basement membrane adhesion with a chaotropic salt solution (2 M NaSCN), which produced intact, full thickness tissue sheets. Extended extraction of the cells and sheets with an alkaline urea buffer followed by analysis of these extracts on denaturing polyacrylamide gels revealed that both preparations contained six major polypeptides in the mol wt range of 50,000-60,000 daltons. Differential solubilization indicated that these proteins are keratin subunits, and amino acid analysis confirmed this for the 53,000-dalton polypeptide (53K) purified from preparative sodium dodecyl sulfate gels. On two-dimensional gel electrophoresis, 53K produced a group of three acidic spots (alpha, beta, and gamma), with isoelectric points of 4.6, 5.5, and 5.4, respectively. Partial peptide maps revealed that these spots are isoelectric variants of the same protein. Immunization of rabbits with 53K produced a keratin antiserum that was primarily directed against the immunogen and cross-reacted weakly with a 50,000- and a 57,000-dalton band. This work demonstrated the feasibility of obtaining well defined molecular probes from a hormone-responsive epithelial system.

Changes in keratin expression during the estrogen-mediated differentiation of rat vaginal epithelium.

While vaginal cornification occurs naturally during the estrous cycle of the adult rat, it can also be artificially induced by administering exogenous hormone to ovariectomized animals. When the maturation of rat vaginal epithelium was controlled with a timed course of estradiol (E2) injections, total cell extracts from that tissue revealed that six major keratins exhibited stage-specific increases during a 48-h period of stratification and cornification. One-dimensional polyacrylamide gel electrophoresis and electrophoretic immunoblotting showed that the expression pattern involved an earlier rise of the smaller (50,000, 51,000, and 53,000 mol wt) keratins and a later rise of the larger (57,000, 58,000, and 60,000 mol wt) keratins. Polypeptide levels were quantitated by gel densitometry, and an immunodot assay was developed to specifically measure keratin expression as a function of estrogen agonist activity. By way of comparison, four other estrogenic compounds were also evaluated after their administration under a 48-h double injection protocol. Under these circumstances, diethylstilbestrol provided a more potent stimulus than E2, while estriol, enclomiphene and zuclomiphene each produced a pattern of keratin expression and intensity levels that resembled that 24-h stage of E2 treatment. Although the same keratins were expressed by these different compounds, their synthesis was selective, since they often increased 10- to 20-fold while total protein levels only doubled. Keratin levels were correlated with the various degrees of epithelial growth and differentiation observed in parallel histological studies. These results indicate that the expression of vaginal keratins provide a useful model system for the study of estrogen action.

Effects of copper on cytoplasmic and nuclear binding of 17 beta-estradiol in the rat uterus.

Interference of Cu++ with the initial events in estrogen action was tested by determining Cu++ effects on estradiol-receptor interactions. When immature rat uteri were incubated in vitro with [3H] estradiol ([3H]E2), steroid was bound in cytoplasmic fractions and rapidly accumulated in the nuclear fraction in a manner which was dependent upon time and hormone concentration. Uteri which were preincubated with 2 X 10(-4) M CuCl2 for 40-60 min and then exposed to [3H]E2 were found to have a 30-50% decrease in the amount of steroid bound in the cytoplasmic and nuclear fractions. When copper-treated uteri were exposed to [3H]E2 for variable times, the quantity of steroid bound in the cytoplasmic fraction was markedly depressed and the rate of nuclear accumulation of [3H]E2 was significantly decreased. These results show that Cu++ can inhibit [3H]E2 binding to tissue cytoplasmic receptors in vitro and thereby interfere with hormone delivery to target cell nuclei.