To facilitate this study an in vitro human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)2D3 (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)2D3 showed that 1,25(OH)2D3 increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)2D3 promoted the process of osteoblast differentiation and maturation.
This study also indicated that 1,25(OH)2D3 stimulated the de novo synthesis of ALP activity in TE85 cells by the following evidence: 1) Treatment of cellular membranes with 1,25(OH)2D3 did not directly activate pre-existing ALP activity, 2) a brief pre-treatment with 1,25(OH)2D3 (1 hour) was sufficient to stimulate ALP activity 47 hours later, 3) 5,6-Dicloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cycloheximide, inhibitors of transcription and protein translation, respectively, each blocked the stimulation of ALP activity by 1,25(OH)2D3, and 4) the stimulation of ALP activity by 1,25(OH)2D3 was accompanied by a corresponding increase in steady state level of ALP mRNA, showing a temporal cause-and-effect relationship between the two parameters.
Evaluations of the mechanistic action of 1,25(OH)2D3 showed that the increase in the steady state level of ALP mRNA was a result of both an increased rate of ALP gene transcription (measured by a nuclear run-off assay) and by a posttranscriptional increase in ALP mRNA stability. The increase in ALP mRNA stability was shown to depend not on a continuous presence of 1,25(OH)2D3 on the cells, but rather on nascent protein synthesis (i.e., cycloheximide blocked the increase in ALP mRNA stability). This finding suggested that increased ALP mRNA stability was mediated through a de novo synthesis of a 1,25(OH)2D3-inducible protein, which has been tentatively called the \"ALP mRNA stabilizing factor\".
Based on the findings in this study, it is proposed that 1,25(OH)2D3 stimulates human ALP activity through a complex set of mechanisms, involving both transcriptional and posttranscriptional events. Although there is a precedence for the regulation of gene expression by steroid hormones through modulation of both gene transcription and mRNA stability, this is the first time that 1,25(OH)2D3 has been shown to regulate gene expression, or affect the state of osteoblast differentiation, through a combination of mechanisms.
","abstract_html":"<p>To facilitate this study an <em>in vitro</em> human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)<sub>2</sub>D<sub>3</sub> (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)<sub>2</sub>D<sub>3</sub> showed that 1,25(OH)<sub>2</sub>D<sub>3</sub> increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)<sub>2</sub>D<sub>3</sub> promoted the process of osteoblast differentiation and maturation.</p> <p>This study also indicated that 1,25(OH)<sub>2</sub>D<sub>3</sub> stimulated the <em>de novo</em> synthesis of ALP activity in TE85 cells by the following evidence: 1) Treatment of cellular membranes with 1,25(OH)<sub>2</sub>D<sub>3</sub> did not directly activate pre-existing ALP activity, 2) a brief pre-treatment with 1,25(OH)<sub>2</sub>D<sub>3</sub> (1 hour) was sufficient to stimulate ALP activity 47 hours later, 3) 5,6-Dicloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cycloheximide, inhibitors of transcription and protein translation, respectively, each blocked the stimulation of ALP activity by 1,25(OH)<sub>2</sub>D<sub>3</sub>, and 4) the stimulation of ALP activity by 1,25(OH)<sub>2</sub>D<sub>3</sub> was accompanied by a corresponding increase in steady state level of ALP mRNA, showing a temporal cause-and-effect relationship between the two parameters.</p> <p>Evaluations of the mechanistic action of 1,25(OH)<sub>2</sub>D<sub>3 </sub>showed that the increase in the steady state level of ALP mRNA was a result of both an increased rate of ALP gene transcription (measured by a nuclear run-off assay) and by a posttranscriptional increase in ALP mRNA stability. The increase in ALP mRNA stability was shown to depend not on a continuous presence of 1,25(OH)<sub>2</sub>D<sub>3</sub> on the cells, but rather on nascent protein synthesis (i.e., cycloheximide blocked the increase in ALP mRNA stability). This finding suggested that increased ALP mRNA stability was mediated through a <em>de novo</em> synthesis of a 1,25(OH)<sub>2</sub>D<sub>3</sub>-inducible protein, which has been tentatively called the "ALP mRNA stabilizing factor".</p> <p>Based on the findings in this study, it is proposed that 1,25(OH)<sub>2</sub>D<sub>3</sub> stimulates human ALP activity through a complex set of mechanisms, involving both transcriptional and posttranscriptional events. Although there is a precedence for the regulation of gene expression by steroid hormones through modulation of both gene transcription and mRNA stability, this is the first time that 1,25(OH)<sub>2</sub>D<sub>3</sub> has been shown to regulate gene expression, or affect the state of osteoblast differentiation, through a combination of mechanisms.</p>","abstract_has_math":false,"creators":["Kyeyune-Nyombi, Eru"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":["Donna Dee Strong","K-H William Lau","Thomas A. Linkhart","John R. Farley","R. Bruce Wilcox","E Clifford Herrmann"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":1991,"date_issued":"1991-03-01T08:00:00Z","date_published":"1991-03-01T08:00:00Z","updated_at":"2026-07-24T02:54:01Z","subjects":["Biochemical Phenomena, Metabolism, and Nutrition","Biochemistry","Genetic Phenomena","Laboratory and Basic Science Research","Osteopathic Medicine and Osteopathy","Calcitriol -- analysis; Alkaline Phosphatase -- analysis; Osteoblasts -- metabolism; Bone Development."],"languages":["English"],"rights":["This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://scholarsrepository.llu.edu/etd/1490","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Donna Dee Strong","K-H William Lau","Thomas A. Linkhart","John R. Farley","R. Bruce Wilcox","E Clifford Herrmann"]},{"key":"dc:creator","label":"Author","values":["Kyeyune-Nyombi, Eru"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Biochemical Phenomena, Metabolism, and Nutrition","Biochemistry","Genetic Phenomena","Laboratory and Basic Science Research","Osteopathic Medicine and Osteopathy","Calcitriol -- analysis; Alkaline Phosphatase -- analysis; Osteoblasts -- metabolism; Bone Development."]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["English"]},{"key":"dc:rights","label":"Dc Rights","values":["This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarsrepository.llu.edu/etd/1490"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["To facilitate this study an in vitro human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)2D3 (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)2D3 showed that 1,25(OH)2D3 increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)2D3 promoted the process of osteoblast differentiation and maturation.
This study also indicated that 1,25(OH)2D3 stimulated the de novo synthesis of ALP activity in TE85 cells by the following evidence: 1) Treatment of cellular membranes with 1,25(OH)2D3 did not directly activate pre-existing ALP activity, 2) a brief pre-treatment with 1,25(OH)2D3 (1 hour) was sufficient to stimulate ALP activity 47 hours later, 3) 5,6-Dicloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cycloheximide, inhibitors of transcription and protein translation, respectively, each blocked the stimulation of ALP activity by 1,25(OH)2D3, and 4) the stimulation of ALP activity by 1,25(OH)2D3 was accompanied by a corresponding increase in steady state level of ALP mRNA, showing a temporal cause-and-effect relationship between the two parameters.
Evaluations of the mechanistic action of 1,25(OH)2D3 showed that the increase in the steady state level of ALP mRNA was a result of both an increased rate of ALP gene transcription (measured by a nuclear run-off assay) and by a posttranscriptional increase in ALP mRNA stability. The increase in ALP mRNA stability was shown to depend not on a continuous presence of 1,25(OH)2D3 on the cells, but rather on nascent protein synthesis (i.e., cycloheximide blocked the increase in ALP mRNA stability). This finding suggested that increased ALP mRNA stability was mediated through a de novo synthesis of a 1,25(OH)2D3-inducible protein, which has been tentatively called the \"ALP mRNA stabilizing factor\".
Based on the findings in this study, it is proposed that 1,25(OH)2D3 stimulates human ALP activity through a complex set of mechanisms, involving both transcriptional and posttranscriptional events. Although there is a precedence for the regulation of gene expression by steroid hormones through modulation of both gene transcription and mRNA stability, this is the first time that 1,25(OH)2D3 has been shown to regulate gene expression, or affect the state of osteoblast differentiation, through a combination of mechanisms.
"]},{"key":"dc:title","label":"Title","values":["Molecular Mechanism of the Stimulation of Alkaline Phosphatase Activity in Human Bone Cells by 1,25(OH)2 D3"]}]}],"canonical_facts":{"dc:contributor":["Donna Dee Strong","K-H William Lau","Thomas A. Linkhart","John R. Farley","R. Bruce Wilcox","E Clifford Herrmann"],"dc:creator":["Kyeyune-Nyombi, Eru"],"dc:description.abstract":["To facilitate this study an in vitro human model system was established that exhibited many aspects of normal osteoblasts. The human osteosarcoma cell line (TE85 cells) expressed a skeletal alkaline phosphatase activity (an accepted bone cell differentiation marker) which was stimulated by 1,25(OH)2D3 (a potent differentiating agent), under serum-free conditions in a dose-dependent, time-dependent, and cell density-dependent manner. Cytochemical analysis of the stimulation of ALP activity by 1,25(OH)2D3 showed that 1,25(OH)2D3 increased the number of TE85 cells that expressed detectable ALP activity, suggesting that 1,25(OH)2D3 promoted the process of osteoblast differentiation and maturation.
This study also indicated that 1,25(OH)2D3 stimulated the de novo synthesis of ALP activity in TE85 cells by the following evidence: 1) Treatment of cellular membranes with 1,25(OH)2D3 did not directly activate pre-existing ALP activity, 2) a brief pre-treatment with 1,25(OH)2D3 (1 hour) was sufficient to stimulate ALP activity 47 hours later, 3) 5,6-Dicloro-1-β-D-ribofuranosylbenzimidazole (DRB) and cycloheximide, inhibitors of transcription and protein translation, respectively, each blocked the stimulation of ALP activity by 1,25(OH)2D3, and 4) the stimulation of ALP activity by 1,25(OH)2D3 was accompanied by a corresponding increase in steady state level of ALP mRNA, showing a temporal cause-and-effect relationship between the two parameters.
Evaluations of the mechanistic action of 1,25(OH)2D3 showed that the increase in the steady state level of ALP mRNA was a result of both an increased rate of ALP gene transcription (measured by a nuclear run-off assay) and by a posttranscriptional increase in ALP mRNA stability. The increase in ALP mRNA stability was shown to depend not on a continuous presence of 1,25(OH)2D3 on the cells, but rather on nascent protein synthesis (i.e., cycloheximide blocked the increase in ALP mRNA stability). This finding suggested that increased ALP mRNA stability was mediated through a de novo synthesis of a 1,25(OH)2D3-inducible protein, which has been tentatively called the \"ALP mRNA stabilizing factor\".
Based on the findings in this study, it is proposed that 1,25(OH)2D3 stimulates human ALP activity through a complex set of mechanisms, involving both transcriptional and posttranscriptional events. Although there is a precedence for the regulation of gene expression by steroid hormones through modulation of both gene transcription and mRNA stability, this is the first time that 1,25(OH)2D3 has been shown to regulate gene expression, or affect the state of osteoblast differentiation, through a combination of mechanisms.
"],"dc:identifier":["https://scholarsrepository.llu.edu/etd/1490"],"dc:language":["English"],"dc:rights":["This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights."],"dc:subject":["Biochemical Phenomena, Metabolism, and Nutrition","Biochemistry","Genetic Phenomena","Laboratory and Basic Science Research","Osteopathic Medicine and Osteopathy","Calcitriol -- analysis; Alkaline Phosphatase -- analysis; Osteoblasts -- metabolism; Bone Development."],"dc:title":["Molecular Mechanism of the Stimulation of Alkaline Phosphatase Activity in Human Bone Cells by 1,25(OH)2 D3"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T02:54:01Z"}