Team:SJTU-BioX-Shanghai/project

Project overview: Synthetic-biological Approaches to Osteoarthritis (OA)

Background

The eukaryotic approach

Design

Detector

The first thing we must face in our approaches to OA is how to detect cells and tissues where hypertrophic differentiation takes place. Considering the eukaryotic method will be applied by inserting the curing circuit into the chromosome of mammalian cells, what we need to do is to find out the molecular markers of OA whose expression level will increase in the case of hypertrophic differentiation.

The type X collagen gene (Col10a1) is such a specific molecular marker of hypertrophic chondrocytes. Multiple cis-elements and trans-acting factors have been reported to regulate Col10a1 expression both in vitro and in vivo and studies have been made on the 5’ flanking region of Col10a1 gene. Scientists found out that Col10a1 promoter locates between 4410 upstream of the initial of transcription and 643 downstream. Further studies showed that when six copies of a 90bp fragment from a enhancer site between -4.4kb to -3.8kb is aligned with Col10 basal promoter (-120 to +1 bp), the chimeric promoter showed high tissue specific (high efficiency of initiating transcription in hypertrophic chondrocytes).

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In approaches of SJTU-BioX-Shanghai, we use the 90bp enhancer fragment (2X) and align it with synthetic JeT core promoter. The chimeric promoter we constructed is supposed to have high tissue specificity and it would initiate transcription only when it is transfected into mammalian cells. The tissue specific promoter restricts the expression of our Supervisor device in hypertrophic chondrocytes. And prevent the inserting genes from harming other normal tissues.

Growth differentiation factor 5 (GDF5) is also a molecular maker of chondrocytes and it is known to be involved in joint formation. Studies have been made on the 5’ flanking region of GDF5 gene and researchers found out GDF5 promoter locates between 1101 upstream of the transcription initiating point and 367 downstream.

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As can be seen from Figure 2, the second DNA fragment (which is 769bp long) has the highest luciferase activity. Therefore, we insert our Supervisor device just downstream of the 769bp GDF5 promoter.

Supervisor

After the detection of tissue where OA takes place, the Supervisor begins to work. Its job is to control the expression of the curing system (Actuator) under the instruction of external signals.

Channelrhodopsin-2 (ChR2) is a light gate ion channel serve as sensory photoreceptors in unicellular green algae2. In the project of team SJTU-BioX-Shanghai this year, we use ChR2 (courtesy of Dr. Weidong Li) as a photosensitive calcium channel which introduces calcium signals into the cell when it is exposed to blue light.

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After the Ca2+ enters the cell, the SJTU-BioX-Shanghai team modified cellular signal pathways which cross-talks with calcium signal. As a result, the cells begin to response to external light signal and initiate the transcription of the Actuator device.

We found and modified two cellular pathways which are related to Ca2+. One is calcium dependent T Cell Receptor (TCR) signaling pathway and the other calcium-dependent mitogen-activated protein kinase (MAPK) pathway.

Calcium dependent TCR pathway:

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The gene regulatory network can be characterized via three pathways’ synergic effect on the gene expression with a cascade of interactions between protein modules involved. The key nodes of the network are CAM, MEF2-Cabin1 complex, and MEF2-NFAT complex. (Further discussion of the three pathways’ synergic work will be showed in the modeling part.)

The effect of the cellular signal transduction is showed in figure 4 below:

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In the absence of calcium, Mitogen Enhancer Factor 2 (MEF2) transcription factor binds to specific DNA sequence upstream of core promoter and recruits cytokine Cabin1, who further recruits Histone deacetylase (HDAC) and blocks transcription. While the cell is stimulated with blue light, ChR2 will introduce more Ca2+, and calcium influx will result in the release of Cabin1-mSin3-HDACs complex. Meanwhile, MEF2 will recruit p300 Histone acetyltransferase (HAT) instead. And transcription is initiated at that point.

In the design of team SJTU-BioX-Shanghai, we aligned MEF2 enhancer with synthetic JeT promoter in order to make the curing system under the control of calcium signals.

Calcium dependent MAPK pathway:

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Cellular MAPK pathway is one of the best studied signaling pathways scientists have focused on. The pathway begins from the activation of membrane-bonded Ras protein and through the activation of extracellular signal-regulated kinase (ERK), the extracellular signal is transducted into the nucleus.

While calcium dependent MAPK pathway is mainly studied in brain and it controls synaptic plasticity. The reason for which Ca2+ could cross-talk with MAPK pathway in brain is that the membrane-bonded Ras protein is activated by Ras-GRF1, a Ca2+/calmodulin-dependent Ras-guanine-nucleotide-releasing factor, which is highly brain specific. Ras-GRF1 interact with NMDA subtype of glutamate receptors (NMDAR) and tranduces signals of Ca2+ influx from the ion channel to the activation of Ras protein.

After the MAPK pathway is activated, activated ERK enters the nucleus and further activates transcription factors such as cyclic AMP response element (CRE)-binding protein (CRE-BP) family. (shown in the figure below)

In the design of team SJTU-BioX-Shanghai, we fused ChR2 ion channel and Ras-GRF1 (courtesy of Prof. Grigory Krapivinsky) so that Ras-GRF1 is sensitive to local increase of Ca2+ introduced by ChR2. And in order to detect the signals from MAPK pathway, we build synthetic promoter with CRE enhancer aligned with JeT core promoter.

Actuator

Osteoarthritis (OA) has two major characters, joint matrix is degraded and chondrocytes undergo disordered and hypertrophic differentiation. In order to reverse the hypertrophic differentiation, the team SJTU-BioX-Shanghai comes up with the idea of induced pluripotent stem cells (iPS cells).If we could reverse the hypertrophic chondrocytes into iPS cells, we might be able to have further stimulation on these cells and turn them into normal chondrocytes again. Although most of these designs are based on our hypothesis, we are eager to have them done. When it comes to the degradation of joint matrix, our solution is much simpler. We cloned the gene col2a1, which is used to replenish joint matrix, and make its expression under the control of our Supervisor.

The prokaryotic approach

References

[1] Qiping Zheng et al. Localization of the Cis-Enhancer Element for Mouse Type X Collagen Expression in Hypertrophic Chondrocytes In Vivo. JOURNAL OF BONE AND MINERAL RESEARCH. Volume 24, Number 6, 2009 [2] Yoshinari Miyamoto, Akihiko Mabuchi et al. A functional polymorphism in the 5' UTR of GDF5 is associated with susceptibility to osteoarthritis. NATURE GENETICS. 25 March 2007 [3] Leopoldo Petreanu et al. The subcellular organization of neocortical excitatory connections. NATURE. Vol 457 26 February 2009 [4] Jun O. Liu et al. Cabin1 Represses MEF2-Dependent Nur77 Expression and T Cell Apoptosis by Controlling Association of Histone Deacetylases and Acetylases with MEF2. Immunity. Vol. 13, 85–94, July, 2000 [5] Gareth M. Thomas, Richard L. Huganir. MAPK cascade signalling and synaptic plasticity. NATURE REVIEWS NEUROSCIENCE. VOLUME 5 MARCH 2004 [6] Grigory Krapivinsky, Luba Krapivinsky, Yunona Manasian. et al. The NMDA Receptor Is Coupled to the ERK Pathway by a Direct Interaction between NR2B and RasGRF1. Neuron. Vol. 40, 775–784, November 13, 2003 [7] Charles L. Farnsworth. et al. Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF. NATURE. Aug 10, 1995 [8] Huibin Yang. et al. Phosphorylation of the Ras-GRF1 Exchange Factor at Ser916/898 Reveals Activation of Ras Signaling in the Cerebral Cortex. THE JOURNAL OF BIOLOGICAL CHEMISTRY. Vol. 278, No. 15, Issue of April 11 [9] Adam J. Shaywitz, Michael E. Greenberg CREB: A STIMULUS-INDUCED TRANSCRIPTION FACTOR ACTIVATED BY A DIVERSE ARRAY OF EXTRACELLULAR SIGNALS. ANNU. REV. BIOCHEM. 1999