Effect of hypophysectomy on the proliferation and differentiation of rat bone marrow stromal cells

James K. Yeh1,2, Jodi F. Evans1, Meng-Meng Chen1, and John F. Aloia1,2

1 Department of Medicine, Winthrop-University Hospital, Mineola 11501; and 2 The Health Sciences Center, State University of New York, Stony Brook, New York 11794

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Conditions such as estrogen deficiency, skeletal unloading, and aging have all been demonstrated to have various effects on the proliferation and differentiation of bone marrow stroma-derived osteoprogenitor cells. Here we have sought to examine the effects of pituitary hormone deficiency on the proliferation and the differentiation of these osteoprogenitor cells using the hypophysectomized (HX) rat as a model. In the present study, we use an in vitro culture system to examine the effects of HX on the osteogenic potential of rat bone marrow stroma. With the intact animal as a control, we used [3H]thymidine incorporation and cell number as indexes of proliferation. We also measured alkaline phosphatase enzyme activity, relative levels of osteocalcin expression with RT-PCR, and osteopontin and bone sialoprotein steady-state levels by Northern blot to delineate the effect on differentiation. Our results indicate that osteoprogenitor cells exposed to a pituitary hormone-deficient environment in vivo demonstrate an enhanced proliferative capacity and also exhibit an augmented expression of differentiation markers when exposed to an optimal environment in vitro.

pituitary hormone; osteoblasts; osteogenesis; alkaline phosphatase; calcium

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE SKELETON OF VERTEBRATES is capable of extensive regeneration (modeling and remodeling), which serves to maintain its health and strength. To retain this capacity, it must possess precursor cell populations capable of differentiating into fully functional osteoblasts and osteoclasts, and the quantity of cells in these precursor pools must be maintained through proliferation (5). Results of studies performed on cells from animals are consistent with the hypothesis that the renewal of the osteoblast population at the bone surface is dependent on the recruitment, proliferation, and differentiation of osteoprogenitor cells (18, 31). It has been determined that cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from multipotential mesenchymal stem cells present in the bone marrow stroma (2, 15, 19).

In vitro culture systems have been established in an effort to characterize the stromal system of bone. Conditions permissive for osteoblast differentiation from marrow stromal cells have been tested extensively. These cells, when grown in the presence of beta -glycerophosphate, ascorbic acid, and dexamethasone, have been proven to form mineralized bone nodules (4, 6, 22, 28). Three stages of development have been delineated for these primary derived osteoblasts: proliferation, extracellular matrix (ECM) maturation, and ECM mineralization (2, 26, 28). As the osteoprogenitor develops into the preosteoblast and osteoblast under these culture conditions, it expresses various bone-specific markers in a temporal manner. For example, type I collagen and alkaline phosphatase (AP) expression is relatively high at early stages but then decreases as mineralization occurs; osteopontin (OPN) is the first matrix protein to appear followed by bone sialoprotein (BSP) and osteocalcin (OCN), with BSP detected first and OCN appearing with mineralization (2).

Conditions such as estrogen deficiency, skeletal unloading, and aging have all been demonstrated to have various effects on the proliferation and differentiation of osteoprogenitor cells derived from bone marrow stroma. Here we have sought to examine the effects of pituitary hormone deficiency on the proliferation and the differentiation of bone marrow-derived osteoprogenitor cells with the hypophysectomized (HX) rat as a model. The HX rat has been used as a model for the effects of pituitary hormone deficiency on bone for many years. Systemically, it results in a suppression of weight gain, a reduction in lean mass, and decreased skeletal growth (8, 14, 30, 33). One of the most striking effects of HX demonstrated in vivo is a cessation of longitudinal growth (14, 30, 33). Hypophysectomy has also been shown to result in cancellous bone loss because of the suppression of longitudinal growth-dependent bone gain and the inhibition of tissue-based bone turnover, with more suppression occurring in bone formation than in resorption (8, 33). In cortical bone, HX results in suppressed radial growth-dependent bone gain, which is associated with a decrease in modeling-dependent bone formation (8). Studies focusing on de novo bone differentiation have yielded similar results. Not only does HX markedly reduce and delay osteogenesis, but also the quality of new bone formed is deficient in the absence of hypophysial hormones (21, 29). On examination of the bone surface of the HX animal, we have discovered a reduction in the osteoblast and osteoclast population compared with an intact age-matched control.

Currently, it is not known whether the reduction in osteogenesis at the bone surface can be attributed to a reduction in the capacity of osteoprogenitors to proliferate and develop into mature osteoblasts. In the present study, we use an in vitro culture system to examine the effects of HX on the osteoprogenitor potential of rat bone marrow stroma. With the intact animal as a control, we used [3H]thymidine incorporation and cell number as indexes of proliferation. We also measured AP enzyme activity, relative levels of OCN expression with RT-PCR, and OPN and BSP steady-state levels by Northern blot to delineate the effect on differentiation. Our results indicate that osteoprogenitor cells exposed to a pituitary hormone-deficient environment in vivo demonstrate an enhanced proliferative and differentiative capacity in vitro when their environment is again optimal.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animal care. Age-matched HX and intact control female, 8-wk-old Sprague-Dawley rats were purchased from Hilltop where the hypophysectomy surgeries were performed. On arrival, 3 days postoperatively, and throughout the experiment, the HX rats were given 3% sucrose water and allowed free access to a standard pelleted chow diet (Rodent Laboratory Chow 5001, Ralston Purina, St. Louis, MO). Animals were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and animal protocols were approved by the Laboratory Animal Care Committee of Winthrop University Hospital.

Histomorphometric analysis. Rats were labeled with 8 mg/kg of calcein subcutaneously (Sigma, St. Louis, MO) at 3 days before being killed. The left and right tibiae were removed and cut with an Isomet saw (Buehler, Lake Bluff, IL). The proximal section of each right tibia was stained by Villanueva osteochrome bone stain (Polysciences, Warrington, PA) for 5 days and then processed for methyl methacrylate embedding without decalcification (33). The specimens were destained and dehydrated with sequential changes (70, 95, and 100%) of ethanol solution and xylene and then embedded in methyl methacrylate (Eastman Organic Chemicals, Rochester, NY). Proximal tibiae were frontal-cut longitudinally with a diamond wire histo-saw (Delaware Diamond Knives, Wilmington, DE) at a thickness of 40 µm and then coverslipped with Eukitt (Calibrated Instruments, Hawthorne, NY).

Histomorphometric parameters of cancellous bone of the proximal tibia were measured with a digitizing morphometry system (OsteoMetrics, Atlanta, GA) and the nomenclature standard (20) as described previously (33). Briefly, the measured parameters included total tissue area, trabecular bone area and perimeters, and calcein labeled perimeters in the secondary spongiosa of the metaphysis in the region 1-4 mm proximal to the growth plate-metaphysial junction. These parameters were then used to calculate cancellous bone volume, total volume, and labeled surface tissue area.

The left tibia was removed and decalcified for 5 wk at 4°C in 10% EDTA containing 7.0% sucrose (pH 7.2). After a thorough wash in PBS solution, the decalcified bones were dehydrated in graded alcohol and embedded in 2-hydroxyethyl methacrylate (JB4-Plus embedding kit, Polysciences). Polymerization was carried out at 4°C in a cold room overnight. Polymerized blocks were kept in a desiccator at 4°C until sectioning. Sections of 4-µm thickness were cut with a microtome (2040 Reichert-Jung, Cambridge Instruments, Buffalo, NY) and then mounted on glass slides. Tartrate-resistant acid phosphatase was localized with a commercially available kit with alpha -naphthyl phosphate disodium salt as substrate (Sigma) (33).

Cell culture. Both femoral and tibial bones were removed from the rats at 6 wk post-HX. Age-matched intact rats served as the control group. Under aseptic conditions, the bones were dissected free of soft tissue, and the epiphysial cartilage was scraped away with a scalpel and discarded. The distal end of the bones was then cut open, and bone marrow was flushed out with 10 ml alpha -MEM medium (without phenol red) supplemented with 10% FBS, antibiotic-antimycotic, 50 µg/ml ascorbic acid, and 2 mM L-glutamine (basal medium) with an 18-gauge needle for the femur and a 22-gauge needle for the tibia. The bone marrow cells were pooled from each of the four bones of each animal, and a single cell suspension was created by repeated flushing through an 18-gauge needle followed by a 22-gauge needle. For the initial proliferation experiments, cells were seeded at a density of 3.5 × 105/cm2 and allowed to attach for 3 days before the removal of nonadherent cells. Differentiation experiments were carried out in the above medium supplemented with 10 mM beta -glycerophosphate and 10-8 M dexamethasone (differentiation medium), and initial plating was at the same density as in the proliferation experiments. Cell numbers were determined with a hemocytometer.

Adherent and nonadherent cell fractions. Bone marrow cells were plated at a density of 3.5 × 105/cm2. After a 1-day initial attachment period, the nonadherent cells in the medium were removed and cultured in separate dishes, and either basal or differentiation medium was replaced in the adherent culture. The first medium change for the adherent cultures took place 4 days later, and medium was changed every second day thereafter. The nonadherent cultures were allowed a 4-day attachment period before the medium was replaced with fresh basal or differentiation medium. Thereafter, the medium was changed every second day. Another set of dishes was seeded at the same density and allowed to attach for 5 days before the nonadherent cells were removed and the medium was replaced with either fresh basal or differentiation medium. Cell number was determined after trypsinization with a hemocytometer.

[3H]thymidine incorporation. Bone marrow cells were plated as stated above and cultured in basal medium. The medium was replaced after 72 h, the adherent cells were cultured for another 4 days, and the medium was changed every 2 days. One microcurie of [3H]thymidine (37 Ci/mmol, Amersham, Arlington Heights, IL) was added to each well, and the cells were cultured for another 24 h. The medium was then removed, and the cells were washed with PBS and detached with 0.05% trypsin in PBS. The cells were collected by centrifugation, lysed with 0.1 N sodium hydroxide, and [3H]thymidine incorporation was measured with a scintillation counter and expressed in counts per minute.

AP activity. On day 14 of culture, the cells were washed with 1× PBS and fixed with ice-cold 95% ethanol (EtOH). The cells were then washed with distilled H2O and allowed to equilibrate in 20 mM bicarbonate buffer, pH 8.8, followed by a 20-min incubation at room temperature with 1 mg/ml p-nitrophenylphosphate in bicarbonate buffer with continuous agitation. p-Nitrophenol in the supernatant was then measured by spectrophotometry at 405 nm, and the results were expressed as nanomoles per minute per 105 cells. Histochemical staining for AP was performed with the Sigma 85L-1 kit according to the directions of the manufacturer. Cell number for the AP cultures was obtained by the method of Currie (9). After the AP activity of the cells was measured, the cells were fixed again in 10% Formalin for further mechanical stability. They were then washed once with tap water and twice with borate buffer (10 mM, pH 8.8), stained with 1% methylene blue in borate buffer for 15 min, and then rewashed several times with tap water followed by borate buffer. Methylene blue-positive colony-forming units (CFU) were used as a measure of total CFU-fibroblast (f) colony numbers. Bound methylene blue was then eluted with 1% HCl, and the absorbance was read at 650 nm. Cell number was then determined by comparison with a standard curve.

Calcium. At day 21 of culture, calcium in the cell layers was extracted with 1% HCl, and calcium content was measured with atomic absorbance. Mineralized nodules were histochemically stained at day 18 of culture with alizarin red, pH 6.2, and nodules were counted manually (24).

RT-PCR for semiquantitation of OCN expression. Bone marrow cells were initially plated at a density of 5 × 105/cm2 in 60-mm dishes and allowed to adhere to the dish for 5 days before the nonadherent cells were removed and the basal medium was replaced with differentiation medium. The cells were then extracted for RNA at 7, 9, 12, 14, and 16 days of culture with Trizol Reagent (Life Technologies) following the recommendations of the manufacturer. RNA (1 µg) was used in each reaction and was first treated with DNase I (Life Technologies). The RNA was then reverse transcribed under the following conditions: 50 mM Tris · HCl (pH 8.3), 75 mM KCl, 20 mM DTT, 3 mM MgCl2, 0.5 mM each dGTP, dATP, dTTP, and dCTP, 100 pg oligo(dT)12-18, 10 U RNase inhibitor (Life Technologies), and 50 U M-MLV reverse transcriptase (Life Technologies). The reaction was incubated at 37°C for 2 h followed by 100°C for 10 min and then immediately placed on ice. Aliquots (10 ml) of the reverse transcription reaction were used in the simultaneous PCR reactions for rat OCN and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reactions were carried out under the following conditions: 20 mM Tris · HCl (pH 8.0), 50 mM KCl, 0.2 mM each dATP, dCTP, dGTP, and dTTP, 2.5 mM MgCl, 1.25 U Taq polymerase (Life Technologies), and 50 pmol each of forward and reverse primers. Primer sequences were as published in Fleet and Hock (12). Reactions were carried out with a Gene-Amp PCR system 2400 thermocycler (Perkin-Elmer, Norwalk, CT). Cycling parameters were as follows: 95°C for 5 min, followed by 20 cycles of 95°C for 15 s, 55°C for 1 min 28 s, 72°C for 45 s, with a final 72°C hold for 7 min. Twenty cycles of PCR were determined to be within the linear range for both OCN and GAPDH.

Quantitation of PCR products and confirmation of sequence identity were performed by Southern blot analysis followed by chemiluminescent detection with a modification of the method of Fleet and Hock (12). Briefly, the Genius nonradioactive detection kit (Boehringer Mannheim, Indianapolis, IN) was utilized with slight modification. The PCR reactions were separated on 2.5% agarose gels. After electrophoresis, the gels were denatured, neutralized, and transferred to Biodyne B membrane (Life Technologies) with 10× standard sodium citrate (SSC) overnight. DNA was fixed to the membrane by baking at 80°C for 1 h. Membranes were prehybridized in 50% formamide, 5× SSC, 5% blocking reagent (Boehringer Mannheim), 0.1% N-laurylsarcosine, and 0.02% SDS for at least 4 h at room temperature. Approximately 15 pmol of 3'-end digoxigenin-linked ddUTP-labeled probe (nonradioactive 3'-end labeling kit from Boehringer Mannheim) per 100 cm2 membrane were added to the prehybridization buffer and allowed to hybridize overnight. OCN was hybridized at 31.8°C and GAPDH at 23°C as described in Fleet and Hock (12). The sequences of the nested oligonucleotide probes were 1) rat OCN, 5'-GTCTATTCACCACCTTACTGCCCTC-3' and 2) rat GAPDH, 5'-CTAAGCAGTTGGTGGTGCA-3' (Midland Certified Reagent). After hybridization, the membranes were washed twice at room temperature in 2× SSC-0.1% SDS for 30 min followed by one 15-min wash in 0.1× SSC-0.1% SDS. Detection of the digoxigenin probe was carried out at room temperature. Membranes were washed in buffer 1 (0.15 M NaCl and 0.1 M maleic acid, pH 7.5) for 5 min followed by a 1-h incubation in buffer 2 (buffer 1 with 1% blocking reagent). A 1:5,000 dilution of the anti-digoxigenin-AP conjugate was then made in buffer 2, and the membrane was incubated in this solution for 30 min followed by two washes, 15 min each, in buffer 1 and a 5-min wash in buffer 3 (0.1 M Tris, 0.15 M NaCl, and 50 mM MgCl2, pH 9.5). A final incubation in a 1:100 dilution of CDP-Star in buffer 3 for 5 min was performed, and the membranes were exposed to Fuji NIF film for 25 s to 5 min. Band intensities were measured with the SigmaGel computer program (Jandel Scientific Software, San Rafael, CA) and an HP Desk Scan II scanner. OCN expression was normalized to GAPDH.

Northern analysis. Total cellular RNA was extracted from cultures as stated above, and 15-µg aliquots were fractionated on 1.2% agarose-formaldehyde gels. The RNA was then transferred to Biodyne B membrane (Life Technologies) with 20× SSC overnight. Membranes were prehybridized in Hybrisol I (Oncor, Gaithersburg, MD) and 0.5 mg/ml sonicated salmon sperm DNA at 42°C for >2 h. The following cDNA probes were used: a 1.0-kb mouse OPN EcoR I fragment, a 1.0-kb mouse BSP EcoR I fragment (generous gifts of Dr. David Rowe, University of Connecticut, Farmington, CT), and a 1.2-kb EcoR I human GAPDH fragment (Clonetech, Palo Alto, CA). Probes were labeled with [32P]dCTP (NEN, Boston, MA) with the RadPrime DNA labeling system (Life Technologies). Membranes were hybridized overnight at 42°C followed by two room temperature washes in 2× SSC-0.1% SDS for 20 min each, one room temperature wash with 0.2× SSC-0.1% SDS for 15 min, and a final wash with 0.2× SSC-0.1% SDS at 42°C for 15 min. The membranes were then exposed to Fuji NIF film overnight with intensifying screens at -80°C. Between hybridizations, bound probes were removed with 0.1% SDS at 80°C. Band intensities were measured with the SigmaGel computer program (Jandel Scientific Software) and an HP Desk Scan II scanner. Expression levels were normalized to GAPDH expression.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Body weight and histomorphometric data. At 6-wk postsurgery, as previously demonstrated, the body weight of the HX rats remained within a 5% range of their initial presurgery weight, whereas the age-matched intact control rats experienced a 32% gain in weight (Table 1). The cancellous bone volume of the proximal tibia was significantly decreased by 6-wk post-HX compared with the intact control group. Using calcein labeling to examine the osteoblast population on the bone surface, we found that the osteoblast number was decreased by HX. Furthermore, the osteoclast number per millimeter of growth plate was also decreased to less than one-half of that of the intact animals.

                              
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Table 1.   Effect of hypophysectomy on body weight and histomorphometric indexes measured in tibial metaphysis

Characterization of bone marrow stromal cell proliferation under basal and differentiating conditions. Total marrow stromal cells recovered from the HX animals were significantly less than total numbers recovered from the intact animals (3.35 ± 0.57 × 108 cells HX vs. 4.57 ± 0.77 × 108 cells control). These data represent the total number of cells recovered from two femora and two tibiae (n = 10). However, initial plating densities were the same in both groups throughout the experiment. After 7 days of incubation in basal medium, the 24-h [3H]thymidine incorporation was 88% greater in the HX group, but the cell number per dish did not differ from the intact control (Table 2). However, by 14 days of culture in basal medium, the cell number and total CFU-f were both higher (81 and 70%, respectively) in the HX group compared with the intact control. When the initial 3-day attachment period was followed by the addition of differentiation medium, cell number was still greater in the HX group. By day 8 under differentiating conditions, it was already 67% more than the intact control group, and by day 14 it was 90% greater (Table 3). CFU-AP and CFU-f colony numbers, after 14 days of incubation in differentiation medium, were also greater in the HX vs. intact control group by 58 and 19%, respectively. The percentage of AP-positive colonies was also greater in the HX group. Sixty percent of the total colonies in control cultures were AP positive, whereas 80% of the total colonies were AP positive in the HX cultures.

                              
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Table 2.   Proliferation characteristics of bone marrow stromal cells from intact control and HX rats under basal culture conditions

                              
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Table 3.   Proliferation characteristics of bone marrow stromal cells from intact control and HX rats under differentiation culture conditions

Recruitment of noncommitted precursor cells. To investigate whether the increased cell number in the HX group is because of an enhanced recruitment of noncommitted precursors, we separated the stromal cells into adherent and nonadherent fractions. The cell fractions were separated and cultured as stated in MATERIALS AND METHODS. Cell numbers were counted at days 8 and 14 in the basal cultures and at day 14 for cultures grown in differentiation medium. At day 8 of basal culture, the cell number in the 1-day adherent fraction of the HX group was only 44% of the intact control, but by day 14 there was no significant difference in cell number (Fig. 1). This implies that the initial committed mesenchymal precursor population in the HX animals is less than that of the intact control, but after exposure to optimal culture conditions, the HX cells surpass the controls in proliferative capacity. In the nonadherent fractions by day 14, the HX group had a 35% greater cell number than the intact control. Moreover, the HX cultures had greater cell numbers at both time points when cells were allowed to attach and recruit other noncommitted mesenchymal precursor cells for 5 days. Therefore, we conclude that there is an enhanced recruitment of noncommitted stromal cells in the HX cultures, and the proliferative capacity of the committed precursor population surpasses control levels after 14 days of optimal culture conditions. In differentiation medium, the results were similar to those with the basal medium in the 5-day adherent fractions, but in the 1-day adherent and nonadherent fractions, the HX cultures had a much higher cell number (+268 and +218%, respectively) than the intact control cultures. This is possibly an indication that both the committed and noncommitted stromal cells of the HX animals are more responsive to differentiating conditions.


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Fig. 1.   Effect of hypophysectomy (HX) on proliferation of committed and noncommitted stromal cells under basal and differentiating conditions. Cell fractions were separated and cultured in basal (bas) or differentiation (diff) medium as described in MATERIALS AND METHODS, and cell numbers were measured at days 8 and 14. Values are means ± SD of 3 replicate cultures from each of 6 different animals/group. Hypox, hypophysectomy; adh, adherent; nadh, nonadherent. * Significant difference between HX and intact control cultures as determined by Student's t-test.

Characterization of bone marrow stroma-derived osteoprogenitor cell differentiation. CELLULAR INDEXES. When AP activity was measured at day 14 of culture under differentiating conditions, the HX cultures demonstrated a greater activity (at least +110%) in all cell fractions (Fig. 2). Mineralized nodules were counted at day 18, and calcium content was measured at day 21 of incubation. These differentiation parameters were also greater in the HX cultures vs. the intact control cultures in all fractions analyzed. The most significant difference was found with the 1-day adherent fraction, where the intact control cultures had developed <10% the number of nodules and only 15% of the level of calcium as the HX group (Fig. 3). Again these results indicate that both the committed preosteoblasts and the noncommitted stromal cells in the HX cultures have an enhanced sensitivity to differentiating conditions.


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Fig. 2.   Effect of HX on alkaline phosphatase activity on both committed and noncommitted osteoblasts. Cell fractions were separated and cultured in differentiation medium as described in MATERIALS AND METHODS. Alkaline phosphatase activity was measured on day 14 of culture. Open bars, intact control cultures; filled bars, HX cultures. Values are means ± SD of 3 replicate cultures from each of 6 different animals/group. * Significant difference between HX and intact control cultures as determined by Student's t-test.


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Fig. 3.   Effect of HX on mineralization of both committed and noncommitted osteoblasts. Cell fractions were separated and cultured in differentiation medium as stated in MATERIALS AND METHODS. A: cultures were stained with alizarin red at day 18 of culture, and colony-forming units staining positive for calcium (CFU-Ca+) were counted manually. B: cultures were extracted for calcium with 1% HCl at day 21 of culture, and calcium was measured with atomic absorption. Open bars, intact control cultures; filled bars, HX cultures. * Significant difference between intact control and HX cultures as determined by Student's t-test.

MOLECULAR INDEXES. Once we found that the cellular markers of osteoblast differentiation were enhanced in the HX bone marrow stromal cell cultures, we sought to examine a few osteogenic markers to determine whether they too would be enhanced. RT-PCR was used to compare relative levels of OCN mRNA levels. Initially, we analyzed OCN expression levels in 1-day adherent and 5-day adherent cultures after 12 days of incubation. The 12-day time point was used because it has been demonstrated to be near the beginning of matrix maturation and the point at which OCN expression can first be detected in stromal cell-derived osteoblast cultures (16, 32). In both the 1-day and 5-day adherent cultures, the HX group demonstrated higher relative OCN expression at day 12 (3× and 1.7×, respectively) (Fig. 4A). The nonadherent fractions were not analyzed for OCN expression. The significant difference in OCN expression between the control and HX cultures prompted us to investigate temporal differences in expression. After a 5-day initial seed, total RNA was extracted from HX and intact control cultures at days 7, 9, 12, 14, and 16 and analyzed for relative OCN expression (Fig. 4B). No significant difference was seen until day 12, and by day 16, the difference had increased to the point at which HX culture OCN mRNA levels were 2.7 times those of the intact control cultures.


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Fig. 4.   Effect of HX on osteocalcin (OC) expression under differentiation conditions as measured by RT-PCR. Relative expression levels were quantitated with Southern blot followed by nonradioactive detection. A: representative Southern blot demonstrating the effect of HX on OC expression in bone marrow stromal cells subjected to different seeding times. Cells were allowed to attach for either 1 day or 5 days before nonadherent cells were removed and basal medium was replaced with differentiation medium. Cultures were incubated for 12 days before total cellular RNA was extracted. B: densitometric evaluation of OC expression in both intact control and HX cultures after 7, 9, 12, 14, and 16 days of incubation. Stromal cells were allowed to attach for 5 days before nonadherent cells were removed and basal medium was replaced with differentiation medium. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C, control.

Duplicate cultures were also analyzed by Northern blot to compare steady-state expression levels of OPN and BSP. Levels of OPN expression at day 9 were already 1.5 times intact control levels in the HX cultures (Fig. 5A); BSP levels, however, did not surpass the control until day 12, when they reached a level 3.2 times greater than the intact control cultures (Fig. 5B). In the HX cultures, expression levels of both of these markers seem to reach peak levels earlier than intact controls, but, by day 16, there is no significant difference between expression levels. Figure 5C is a representative Northern blot depicting steady-state levels of OPN and BSP transcripts in the HX and intact control cultures at days 12 and 14.


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Fig. 5.   Effect of HX on the osteogenic markers osteopontin (OP) and bone sialoprotein (BSP) under differentiating conditions as measured by Northern blot. A and B: densitometric evaluation of OPN and BSP expression in HX (filled bars) and intact control cultures (open bars) after 9, 12, 14 and 16 days of incubation. Stromal cells were allowed a 5-day attachment period before the nonadherent cells were removed and the basal medium was replaced with differentiation medium. C: representative Northern blot of OPN and BSP expression in HX and intact control cultures at days 12 and 14.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Hypophysectomy is known to result in osteopenia, and the histomorphometric data from these experiments confirm previous conclusions that this osteopenia is, in part, due to a decrease in the osteoblast population on the bone surface (23, 33). Hypophysectomy has also been demonstrated to have a negative effect on hematopoiesis and to result in anemia, leukopenia, thrombocytopenia, and impaired DNA and RNA synthesis in the bone marrow (17). The total number of marrow stromal cells recovered from the HX animals is in parallel with these findings. Total stroma cells recovered from HX animals were significantly less than totals recovered from the intact controls. This study was designed to determine whether or not the reduction in osteogenesis at the bone surface is due to a reduction in the capacity of osteoprogenitors present in the bone marrow to proliferate and develop into mature osteoblasts.

In our initial proliferation studies using a 3-day attachment period, [3H]thymidine incorporation in the HX cultures is significantly greater than in the intact control cultures, but there is no significant difference in cell number at day 8 of culture under nondifferentiating conditions. By day 14, however, the HX group has a significantly greater cell number than the control, demonstrating that the HX committed mesenchymal precursor population, when exposed to an optimal microenvironment in vitro, shows a proliferative capacity that surpasses that of the intact animal. Under differentiating conditions and a 3-day initial attachment period, the HX cultures show greater cell numbers after only 8 days of culture, indicating a possibly greater sensitivity to dexamethasone. When examining total CFU-f under nondifferentiating and differentiating conditions, we again found significantly greater numbers in the HX cultures; when comparing the percentage of total fibroblastic colonies positive for alkaline phosphatase we also found a greater percentage in the HX cultures vs. the control. This implies that the osteoblastic precursors of the HX marrow are a definite contributor to the enhanced proliferative capacity of the marrow stroma. The HX stromal cells also appear to have superior recruitment potential, as evidenced by the greater total number of CFU-f colonies under both conditions. Through visual observation, we also noted that the area of CFU-f and CFU-AP per dish was also greater in the HX cultures, which supports our hypothesis of enhanced proliferative capacity of both mesenchymal precursors and the preosteoblast population.

To investigate the recruitment potential of the two groups we separated the bone marrow stromal cells into adherent and nonadherent fractions as described in MATERIALS AND METHODS and cultured them under nondifferentiating and differentiating conditions. Under nondifferentiating conditions, we discovered that the initial committed mesenchymal precursor population (1-day adherent fraction) of the HX animal appears to be reduced compared with the intact control. However, after 14 days of optimal culture conditions, this fraction of cells from the HX cultures has greater cell numbers, indicating a greater proliferative capacity. The proportion of nonadherent cells in the two groups cannot be deduced in this manner because other factors like recruitment potential contribute to final cell numbers. We also did not measure the proportion of cells in the different fractions and therefore cannot attribute greater cell numbers in the nonadherent fraction of the HX group to greater starting numbers. If our assumption that the initial committed fraction of cells is less in the HX group than when comparing the 5-day adherent fractions, we can say that the committed progenitor population of the HX animal possesses recruitment potential superior to the intact counterparts.

We have also demonstrated that there is a greater induction of the osteoblast phenotype from the noncommitted progenitor population in the marrow stroma by separating these two fractions of progenitor cells and culturing them in differentiation medium. Under these conditions, all fractions from the HX animal showed greater proliferative and differentiation capacities than the intact control cultures. By 14 days of culture in differentiation medium, cell number in the HX cultures was very significantly greater in all fractions, indicating an extreme sensitivity of these cells to differentiating conditions. After examining differentiation markers at the cellular level in the different fractions, we found that AP activity per cell was greater in all fractions of the HX cultures, confirming a greater sensitivity of these cells to differentiating conditions. Because AP activity is not exclusively a marker of osteoblasts, we examined calcium-containing colony-forming units (CFU-Ca) colony numbers and total calcium content as well. These measurements are indicators of mineralization potential. CFU-Ca colony numbers were significantly greater, and calcium content was at significantly increased levels, in all fractions of the HX cultures. These data correlate with the increases in cell number and AP activity of these fractions under differentiating conditions compared with the intact control. Several factors could be at work here. First, the nonadherent and initial adherent progenitors from the pituitary-deficient animal appear to be more sensitive to the inductive properties of the dexamethasone in the medium. Second, the recruitment potential of the adherent precursor population could be augmented as discussed earlier. It has been hypothesized that when the adherent fraction of precursors is exposed to anabolic stimuli present in the culture medium, they secrete factors such as prostaglandin E2, which has been shown to mediate the transition between nonadherent and adherent preosteoblast cells (24, 25). Because the adherent preosteoblasts from the HX animals have an enhanced response to anabolic stimuli as demonstrated through our proliferation experiments, perhaps they are secreting greater amounts of recruiting factors into the medium and therefore augmenting osteoprogenitor recruitment.

Our examination of molecular markers of differentiation has revealed an earlier progression toward mineralization and enhanced levels of expression in HX cultures. We examined gene expression of two noncollagenous matrix proteins, OPN and BSP. OPN expression has been detected during both the proliferative and differentiation stages of osteoblast development (2, 28, 32), but the role it plays in mineralization has not been clearly defined (32). It has been speculated, however, that its function during the proliferative stage of development is related to the mediation of cell attachment and it conditions the surface of the culture dish in preparation for mineralization (28, 32). As in these other osteoblastic development models, we have also detected this matrix protein during the proliferation stage with considerably higher levels detected in the HX cultures. The second matrix protein of which gene expression levels were measured, BSP, has been implicated as an influence in the initial formation of mineralized nodules (32). When temporal patterns and peak expression of BSP between the two groups are compared, HX cells not only reach maximal expression earlier than control cells, but peak levels are also greater than those of control cells as well. Using RT-PCR coupled to Southern blot, we also analyzed relative OCN mRNA levels in the two groups. This osteoblastic marker is not only specific to bone but is also highly correlated with the calcification of the ECM (28). By day 12 of culture, there were considerably greater mRNA levels of OCN in the HX cultures, and this difference remained through day 16.

It has been postulated that proliferation is functionally related to the synthesis of the ECM, and the maturation and organization of the ECM contribute to the shutdown of proliferation. This, in turn, promotes the expression of genes that prepare the matrix for mineralization. ECM mineralization or events during the early stages of mineralization are possibly responsible for downregulating genes expressed during ECM maturation and organization (28). Therefore, in this model of osteoblast development, the formation of the ECM is completely related to the stages of differentiation. If this model is applied to the current study, it may help to explain the phenomenon that is occurring in the differentiation of the HX osteoprogenitors. During the first stage, proliferation is elevated, leading to an enhanced deposition of matrix proteins during the ECM maturation stage, as evidenced by increased OPN levels at the end of this stage. This perhaps led to an early shutdown of proliferation and an early and enhanced preparation of the matrix indicated by a temporal shift to the left and greater peak levels of BSP. Ultimately, these events sparked the increased expression levels of OCN and subsequently mineralization. This explanation still leaves us to explain the causative mechanism behind the initial increase in proliferation of the HX osteoblast lineage cells. Why do the in vivo and in vitro results seem in such stark contrast?

The elevated response of the bone marrow stromal cells of the HX animal when placed in vitro could be because of a hypersensitivity of these cells to a factor(s) present in the fetal bovine serum and lacking for an extended period of time in vivo. The most obvious of these is growth hormone (GH), a major systemic growth regulator and mitogenic factor that exerts its effects on bone both directly and indirectly via insulin-like growth factor (IGF)-I (3, 10). Circulating GH interacts with certain receptors located mainly in hepatic tissue, resulting in the stimulation of the synthesis and secretion of IGF-I (1). IGF-I also has paracrine-autocrine properties and is locally secreted by osteoblasts in response to GH (10). Perhaps these osteoprogenitors experience an upregulation of the IGF-I receptor because of the acute lack of GH and, subsequently, the lack of systemic and local IGF-I. It has been demonstrated that human patients with reduced serum levels of IGF-I experienced a compensatory upregulation of lymphocytic IGF-I receptor gene expression in response to low circulating IGF-I levels (11). If this were also true with osteoblasts, it would explain why, when these cells are placed in culture with fetal bovine serum, of which bovine GH is a definite component, they are stimulated to divide at an elevated rate. These receptors initiate the Ras/Raf/MEK/mitogen-activated protein kinase (MAPK) signaling cascade and MAPKs have been indicated as transcriptional regulators of proliferation in osteoblasts (27). This is only one possibility, however, because the HX animal lacks more than just GH. Many other hormones with anabolic properties in bone are also lacking in vivo, such as parathyroid hormone and estrogen. Moreover, GH replacement in HX rats cannot completely restore trabecular bone formation (7), indicating the involvement of other pituitary and/or local growth factors in bone formation.

In conclusion, we have observed that once bone marrow stromal cells from a HX animal are cultured in vitro, they experience an enhanced proliferative and differentiative capacity compared with those of the intact animal. The cellular and molecular mechanisms behind this phenomenon have yet to be elucidated. An understanding of these regulatory mechanisms is critical for gaining insight into the pathogenesis of pituitary deficiency-related osteoporosis and other bone-related diseases, as well as for the development of new treatment regimens.

    ACKNOWLEDGEMENTS

The authors are grateful to Nancy Ling and Amy Ching-Man Chu for expert technical assistance.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: J. K. Yeh, Metabolism Laboratory, Dept. of Medicine, Winthrop-Univ. Hospital, Mineola, NY 11501.

Received 20 April 1998; accepted in final form 8 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Adamo, M. L., M. A. Bach, C. T. Roberts, and D. LeRoith. Regulation of insulin, IGF-I, and IGF-II gene expression. In: Insulin-Like Growth Factors: Molecular and Cellular Aspects, edited by D. LeRoith. Boca Raton, FL: CRC, 1991, p. 271-303.

2.  Aubin, J. E., F. Liu, L. Malaval, and A. K. Gupta. Osteoblast and chondroblast differentiation. Bone 17, Suppl.: 77S-83S, 1995.

3.   Barnard, R., T. J. Martin, and M. J. Waters. Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH. Endocrinology 128: 1459-1464, 1991[Abstract].

4.   Bellows, C. G., J. E. Aubin, and J. N. M. Heersche. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvarial cells in vitro. Endocrinology 121: 1985-1992, 1987[Abstract].

5.   Beresford, J. N. Osteogenic stem cells and the stromal system of bone and marrow. Clin. Orthop. 240: 270-280, 1989[Medline].

6.   Cheng, S.-L., J. W. Yang, L. Rifas, S.-F. Zhang, and L. V. Avioli. Differentiation of human bone marrow osteogenic stromal cell in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134: 277-286, 1994[Abstract].

7.   Chen, M.-M., J. K. Yeh, and J. F. Aloia. Histologic evidence: growth hormone completely prevents reduction in cortical bone gain and partially prevents cancellous osteopenia in the tibia of hypophysectomized rats. Anat. Rec. 249: 163-172, 1997[Medline].

8.   Chen, M.-M., J. K. Yeh, and J. F. Aloia. Skeletal alterations in hypophysectomized rats. II. A histomorphometric study on tibial cortical bone. Anat. Rec. 241: 513-518, 1995[Medline].

9.   Currie, G. A. Platelet-derived growth factor requirements for in vitro proliferation of normal and malignant cells. Br. J. Cancer 43: 335-343, 1981[Medline].

10.   Ernst, E., and E. R. Froesch. Growth-hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I. Biochem. Biophys. Res. Commun. 151: 142-147, 1988[Medline].

11.   Eshet, R., H. Werner, B. Klinger, A. Silbergeld, Z. Laron, D. LeRoith, and C. T. Roberts. Up-regulation of insulin-like growth factor-I (IGF-I) receptor gene expression in patients with reduced serum IGF-I levels. J. Mol. Endocrinol. 10: 115-120, 1993[Abstract].

12.   Fleet, J. C., and J. M. Hock. Identification of osteocalcin mRNA in non-osteoid tissue of rats and humans by reverse transcription-polymerase chain reaction. J. Bone Miner. Res. 9: 1565-1573, 1994[Medline].

13.   Hu, J. L., X. J. Guan, and E. R. Sanchez. Enhancement of glucocorticoid receptor-mediated gene expression by cellular stress: evidence for the involvement of a heat shock-initiated factor or process during recovery from stress. Cell Stress Chaperones 1: 197-205, 1996.[Medline]

14.   Kember, N. F. Growth hormone and cartilage cell division in hypophysectomized rats. Cell Tissue Kinet. 4: 193-199, 1971[Medline].

15.   Maniatopoulos, C., J. Sodek, and A. H. Melcher. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res. 254: 317-330, 1988[Medline].

16.   Mathieu, E., and J. Merregaert. Characterization of the stromal osteogenic cell line MN7: mRNA steady-state level of selected osteogenic markers depends on cell density and is influenced by 17beta -estradiol. J. Bone Miner. Res. 9: 183-192, 1994[Medline].

17.   Nagy, E., and I. Berczi. Pituitary dependence of bone marrow function. Br. J. Haematol. 71: 457-462, 1989[Medline].

18.   Nijweide, P. J., E. H. Burger, and J. H. Feyen. Cells of bone: proliferation, differentiation and hormonal control. Physiol. Rev. 66: 855-886, 1986[Free Full Text].

19.   Owen, M. Marrow stromal cells. J. Cell Sci. Suppl. 10: 63-76, 1988[Medline].

20.   Parfitt, A. M., M. K. Drezner, F. H. Glorieux, J. A. Kanis, H. Malluche, P. J. Meunier, S. M. Ott, and R. R. Recker. Bone histomorphometry: standardization of nomenclature, symbol, and units. J. Bone Miner. Res. 2: 595-610, 1987[Medline].

21.   Reddi, A. H., and N. E. Sullivan. Matrix-induced endochondral bone differentiation: influence of hypophysectomy, growth hormone, and thyroid-stimulating hormone. Endocrinology 107: 1291-1299, 1980[Abstract].

22.   Rickard, D. J., T. A. Sullivan, B. J. Shenker, P. S. Leboy, and I. Kazhdan. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev. Biol. 161: 218-228, 1994[Medline].

23.   Schmidt, I. U., H. Dobnig, and R. T. Turner. Intermittent parathyroid hormone treatment increases osteoblast number, steady state messenger ribonucleic acid levels for osteocalcin, and bone formation in tibial metaphysis of hypophysectomized female rats. Endocrinology 136: 5127-5134, 1995[Abstract].

24.   Scutt, A., and P. Bertram. Bone marrow cells are targets for the anabolic actions of prostaglandin E2 on bone: induction of a transition from nonadherent to adherent osteoblast precursors. J. Bone Miner. Res. 10: 474-487, 1995[Medline].

25.   Scutt, A., P. Bertram, and M. Brautigam. The role of glucocorticoids and prostaglandin E2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: positive and negative effects. Calcif. Tissue Int. 59: 154-162, 1996[Medline].

26.   Scutt, A., H. Mayer, and E. Wingender. New perspectives in the differentiation of bone forming cells. Biofactors 4: 1-13, 1992[Medline].

27.   Siddhanti, S. R., and L. D. Quarles. Molecular to pharmacologic control of osteoblast proliferation and differentiation. J. Cell. Biochem. 55: 310-320, 1994[Medline].

28.   Stein, G. S., and J. B. Lian. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr. Rev. 14: 424-442, 1993[Medline].

29.   Syftestad, G. T., and M. R. Urist. Growth-hormone dependent matrix-induced heterotropic bone formation. Proc. Soc. Exp. Biol. Med. 163: 411-415, 1980.

30.   Thorngren, K. G., L. I. Hansson, K. Menander-Sellman, and A. Stenstrom. Effect of hypophysectomy on longitudinal bone growth in the rat. Calcif. Tissue Res. 11: 281-300, 1973[Medline].

31.   Wlodarski, K. H. Properties and origins of osteoblasts. Clin. Orthop. 252: 276-293, 1990[Medline].

32.   Yao, K.-L., R. Todescan, and J. Sodek. Temporal changes in matrix protein synthesis and mRNA expression during mineralized tissue formation by adult rat bone marrow cells in culture. J. Bone Miner. Res. 9: 231-240, 1994[Medline].

33.   Yeh, J. K., M.-M. Chen, and J. F. Aloia. Skeletal alterations in hypophysectomized rats. I. A histomorphometric study on tibial cancellous bone. Anat. Rec. 241: 505-512, 1995[Medline].


Am J Physiol Endocrinol Metab 276(1):E34-E42
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