Publication Date

12-2015

Date of Final Oral Examination (Defense)

7-22-2015

Type of Culminating Activity

Thesis

Degree Title

Master of Science in Biology

Department

Biology

Major Advisor

Julia T. Oxford, Ph.D.

Advisor

Cheryl L. Jorcyk, Ph.D.

Advisor

Allan R. Albig, Ph.D.

Abstract

Long bones develop via endochondral ossification, a process in which cartilage precedes bone. During endochondral ossification prechondrogenic cells undergo proliferation and apoptose as cells of the periosteum differentiate into bone. The process of endochondral ossification is regulated by a group of endocrine as well as paracrine molecules that includes bone morphogenetic protein-2 (BMP-2) and parathyroid hormone-related peptide (PTHrP). Collagens are extracellular matrix molecules that are present in a spatiotemporal manner during endochondral ossification. Collagen XI alpha 1 (Col11a1) is a minor fibrillar collagen that is alternatively spliced during development and can result in up to eight different spliceforms. During endochondral ossification Col11a1 spliceforms have a distinct expression profile in the growing long bone. The significance of the different spliceforms has not been characterized. Here, I studied the effects of BMP-2 and PTHrP on the expression of Col11a1 in pre-chondrogenic mouse ATDC5 and pluripotent mouse C2C12 cells. I also investigated the role of different Col11a1 spliceforms in BMP-2-mediated C2C12 osteoblast differentiation. My results suggest a role for BMP-2 in regulating Col11a1 splicing and expression that includes the induction of exons 6A, 7, and 8. Further I show that while PTHrP alone did not affect Col11a1 expression, when combined with BMP-2, PTHrP was able to diminish BMP-2-induced Col11a1 expression. In addition, Col11a1 NTD[p6b-7] reduced BMP-2 activity in C2C12 cells while Col11a1 NTD[p7] did not have any significant effects. Col11a1 NTD[p6b-7] also reduced the expression of alkaline phosphatase and collagen 1 alpha 1 and increased the expression of Runx2 and osteocalcin while NTD[p7-8] induced the expression of osteocalcin, Runx2, and collagen alpha 1. When Col11a1 was knocked down, the expression of BMP-2-induced markers alkaline phosphatase, Runx2, osteocalcin, and collagen 1 alpha 1 were reduced at 24h post induction with BMP-2 suggesting a role for collagen XI alpha 1 during early osteoblast differentiation. In ATDC5 cells, I determined a role for PTHrP in the induction of collagen XI alpha 1 exons 6A, 7, and 8 during early chondrocyte differentiation and exon 6B during late chondrocyte differentiation. Overall the results of this project show a novel role for collagen XI alpha 1 in BMP-2-mediated osteoblast differentiation and also suggest a role for PTHrP in regulating collagen XI alpha splicing and expression during ATDC5 chondrocyte differentiation.

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